Heparanase specific molecular probes and their use in research and medical applications

ABSTRACT

A variety of heparanase specific molecular probes which can be used for research and medical applications including diagnosis and therapy. Specific applications include the use of a heparanase specific molecular probe for detection of the presence, absence or level of heparanase expression; the use of a heparanase specific molecular probe for therapy of a condition associated with expression of heparanase; the use of a heparanase specific molecular probe for quantification of heparanase in a body fluid; the use of a heparanase specific molecular probe for targeted drug delivery; and the use of a heparanase specific molecular probe as a therapeutic agent.

[0001] This is a divisional of U.S. patent application Ser. No.09/704,772, filed Nov. 3, 2000, which is a divisional of U.S. patentapplication Ser. No. 09/322,977, filed Jun. 1, 1999, now U.S. Pat. No.6,531,129, issued Mar. 11, 2003, which is a divisional of U.S. patentapplication Ser. No. 09/071,739, filed May 1, 1998, now U.S. Pat. No.6,177,545, issued Jan. 23, 2001, which is a continuation-in-part of U.S.patent application Ser. No. 08/922,170, filed Sep. 2, 1997, now U.S.Pat. No. 5,968,822, issued Oct. 19, 1999.

FIELD AND BACKGROUND OF THE INVENTION

[0002] The present invention relates to heparanase specific molecularprobes their use in research and medical applications. Moreparticularly, the present invention relates to the use of heparanasespecific molecular probes, such as anti-heparanase antibodies (bothpoly- and monoclonal) and heparanase gene (hpa) derived nucleic acids,including, but not limited to, PCR primers, antisense oligonucleotideprobes, antisense RNA probes, DNA probes and the like for detection andmonitoring of malignancies, metastasis and other non-malignantconditions, efficiency of therapeutic treatments, targeted drug deliveryand therapy.

[0003] Heparan sulfate proteoglycans (HSPGs): HSPGs are ubiquitousmacromolecules associated with the cell surface and extracellular matrix(ECM) of a wide range of cells of vertebrate and invertebrate tissues(1-5). The basic HSPG structure consists of a protein core to whichseveral linear heparan sulfate chains are covalently attached. Thepolysaccharide chains are typically composed of repeating hexuronic andD-glucosamine disaccharide units that are substituted to a varyingextent with N- and O-linked sulfate moieties and N-linked acetyl groups(1-5). Studies on the involvement of ECM molecules in cell attachment,growth and differentiation revealed a central role of HSPGs in embryonicmorphogenesis, angiogenesis, metastasis, neurite outgrowth and tissuerepair (1-5). The heparan sulfate (HS) chains, unique in their abilityto bind a multitude of proteins, ensure that a wide variety of effectormolecules cling to the cell surface (4-6). HSPGs are also prominentcomponents of blood vessels (3). In large vessels they are concentratedmostly in the intima and inner media, whereas in capillaries they arefound mainly in the subendothelial basement membrane where they supportproliferating and migrating endothelial cells and stabilize thestructure of the capillary wall. The ability of HSPGs to interact withECM macromolecules such as collagen, laminin and fibronectin, and withdifferent attachment sites on plasma membranes suggests a key role forthis proteoglycan in the self-assembly and insolubility of ECMcomponents, as well as in cell adhesion and locomotion. Cleavage of HSmay therefore result in disassembly of the subendothelial ECM and hencemay play a decisive role in extravasation of blood-borne cells (7-9). HScatabolism is observed in inflammation, wound repair, diabetes, andcancer metastasis, suggesting that enzymes which degrade HS playimportant roles in pathologic processes.

[0004] Involvement of heparanase in tumor cell invasion and metastasis:Circulating tumor cells arrested in the capillary beds of differentorgans must invade the endothelial cell lining and degrade itsunderlying basement membrane (BM) in order to escape into theextravascular tissue(s) where they establish metastasis (10). Severalcellular enzymes (e.g., collagenase IV, plasminogen activator, cathepsinB, elastase) are thought to be involved in degradation of the BM (10).Among these enzymes is an endo-β-D-glucuronidase (heparanase) thatcleaves HS at specific intrachain sites (7, 9, 11-12). Expression of aHS degrading heparanase was found to correlate with the metastaticpotential of mouse lymphoma (11), fibrosarcoma and melanoma (9) cells.Treatment of experimental animals with heparanase inhibitors (i.e.non-anticoagulant species of low MW heparin) markedly reduced (>90%) theincidence of lung metastases induced by B16 melanoma, Lewis lungcarcinoma and mammary adenocarcinoma cells (8, 9, 13).

[0005] Heparanase activity could not be detected in normal stromalfibroblasts, mesothelial, endothelial and smooth muscle cells derivedfrom non cancerous biopsies and effusions (12). These observationsindicate that heparanase expression may serve as a marker for tumorcells and in particular for those which are highly invasive orpotentially invasive. If the same conclusion can be reached byimmunostaining of tissue specimens, anti-heparanase antibodies may beapplied for early detection and diagnosis of metastatic cell populationsand micro-metastases.

[0006] Our studies on the control of tumor progression by its localenvironment, focus on the interaction of cells with the extracellularmatrix (ECM) produced by cultured corneal and vascular endothelial cells(EC) (14, 15). This ECM closely resembles the subendothelium in vivo inits morphological appearance and molecular composition. It containscollagens (mostly type III and IV, with smaller amounts of types I andV), proteoglycans (mostly heparan sulfate- and dermatansulfate-proteoglycans, with smaller amounts of chondroitin sulfateproteoglycans), laminin, fibronectin, entactin and elastin (13, 14). Theability of cells to degrade HS in the ECM was studied by allowing cellsto interact with a metabolically sulfate labeled ECM, followed by gelfiltration (SEPHAROSE 6B) analysis of degradation products released intothe culture medium (11). While intact HSPG are eluted next to the voidvolume of the column (Kav<0.2, Mr˜0.5×10⁶), labeled degradationfragments of HS side chains are eluted more toward the Vt of the column(0.5<kav<0.8, Mr=5-7×10³) (11).

[0007] Possible involvement of heparanase in tumor angiogenesis:Fibroblast growth factors are a family of structurally relatedpolypeptides characterized by high affinity to heparin (16). They arehighly mitogenic for vascular endothelial cells (EC) and are among themost potent inducers of neovascularization (16, 17). Basic fibroblastgrowth factor (bFGF) has been extracted from subendothelial ECM producedin vitro and from BM of the cornea, suggesting that ECM may serve as areservoir for bFGF (18). Studies on the interaction of bFGF with ECMrevealed that bFGF binds to HSPG in the ECM and can be released in anactive form by HS degrading enzymes (19, 20). Heparanase activityexpressed by platelets, mast cells, neutrophils, and lymphoma cellsreleases active bFGF from ECM and BM (20), suggesting that heparanasemay not only function in cell migration and invasion, but may alsoelicit an indirect neovascular response (18). These results suggest thatthe ECM HSPGs provide a natural storage depot for bFGF and possiblyother heparin-binding growth promoting factors. Displacement of bFGFfrom its storage within ECM may therefore provide a novel mechanism forinduction of neovascularization in normal and pathological situations(6, 18).

[0008] Expression of heparanase by cells of the immune system:Heparanase activity correlates with the ability of activated cells ofthe immune system to leave the circulation and elicit both inflammatoryand autoimmune responses. Interaction of platelets, granulocytes, T andB lymphocytes, macrophages and mast cells with the subendothelial ECM isassociated with degradation of heparan sulfate (HS) by heparanaseactivity (7). The enzyme is released from intracellular compartments(e.g., lysosomes, specific granules) in response to various activationsignals (e.g., thrombin, calcium ionophore, immune complexes, antigens,mitogens), suggesting its regulated involvement and presence ininflammatory sites and autoimmune lesions. Heparan sulfate degradingenzymes released by platelets and macrophages are likely to be presentin atherosclerotic lesions (21). Hence, cDNA probes and anti-heparanaseantibodies may be applied for detection and early diagnosis of theselesions.

[0009] Cloning and expression of the heparanase gene: The cloning andexpression of the human heparanase gene are described in U.S. Pat. No.5,968,822, which is incorporated by reference as if fully set forthherein. A purified fraction of heparanase isolated from human hepatomacells was subjected to tryptic digestion. Peptides were separated byhigh pressure liquid chromatography and micro sequenced. The sequence ofone of the peptides was used to screen data bases for homology to thecorresponding back translated DNA sequence. This procedure led to theidentification of a clone containing an insert of 1020 base pairs (bp)which included an open reading frame of 963 bp followed by 27 bp of 3′untranslated region and a Poly A tail. The new gene was designated hpa.Cloning of the missing 5′ end of hpa cDNA was performed by PCRamplification of DNA from placenta cDNA composite. The plasmidcontaining the entire heparanase cDNA was designated phpa. The joinedcDNA fragment contained an open reading frame which encodes apolypeptide of 543 amino acids with a calculated molecular weight (MW)of 61,192 daltons. The ability of the hpa gene product to catalyzedegradation of heparan sulfate (HS) in vitro was examined by expressingthe entire open reading frame of hpa in High five and Sf21 insect cells,using the Baculovirus expression system. Extracts of infected cells wereassayed for heparanase activity. For this purpose, cell lysates wereincubated with sulfate labeled, ECM-derived HSPG (peak I), followed bygel filtration analysis (SEPHAROSE 6B) of the reaction mixture. Whilethe substrate alone consisted of high molecular weight (MW) material,incubation of the HSPG substrate with lysates of cells infected with hpacontaining virus resulted in a complete conversion of the high MWsubstrate into low MW labeled heparan sulfate degradation fragments.

[0010] In subsequent experiments, the labeled HSPG substrate wasincubated with the culture medium of infected High Five and Sf21 cells.Heparanase activity, reflected by the conversion of the high MW HSPGsubstrate into low MW HS degradation fragments, was found in the culturemedium of cells infected with the pFhpa virus, but not the control pF1virus. Altogether, these results indicate that the heparanase enzyme isexpressed in an active form by cells infected with Baculoviruscontaining the newly identified human hpa gene. In other experiments, wehave demonstrated that the heparanase enzyme expressed by cells infectedwith the pFhpa virus is capable of degrading HS complexed to othermacromolecular constituents (e.g., fibronectin, laminin, collagen)present in a naturally produced intact ECM, in a manner similar to thatreported for highly metastatic tumor cells or activated cells of theimmune system.

[0011] Purification of the recombinant heparanase enzyme: Thepurification of the human heparanase gene are described in U.S. Pat. No.5,968,822, which is incorporated by reference as if fully set forthherein. Sf21 insect cells were infected with pFhpa virus and the culturemedium was applied onto a heparin-SEPHAROSE column. Fractions wereeluted with a salt gradient (0.35-2 M NaCl) and tested for heparanaseactivity and protein profile (SDS/PAGE followed by silver staining).Heparanase activity correlated with the appearance of a protein band ofabout 63 kDa in fractions 19-24, consistent with the expected MW of thehpa gene product. Active fractions eluted from heparin-SEPHAROSE werepooled, concentrated and applied onto a Superdex 75 FPLC gel filtrationcolumn. Aliquots of each fraction were tested for heparanase activityand protein profile. A correlation was found between the appearance of amajor protein of about 63 kDa in fractions 4-7 and heparanase activity.This protein was not present in medium conditioned by controlnon-infected Sf21 cells and subjected to the same purification protocol.

[0012] Research on the involvement of heparanase/HS in tumor cellmetastasis and angiogenesis has been handicapped by the lack ofbiological tools (i.e., molecular probes, antibodies) to explore acausative role of heparanase in disease. U.S. Pat. No. 5,968,822 offers,for the first time, a good opportunity to elucidate the enzyme'sinvolvement in tumor metastasis and angiogenesis and the relateddiagnostic applications.

[0013] On the basis of the examples described below, it appears thatcDNA and RNA probes, PCR primers, and anti-heparanase antibodies(heparanase specific molecular probes) can be applied to detect theheparanase gene and protein and hence for early diagnosis ofmicrometastases, autoimmune lesions, renal failure and atheroscleroticlesions using biopsy specimens, plasma samples, and body fluids.

[0014] Specificity and advantages over other reported antibodies: Avariety of blood, tumor cells and certain normal cells have been shownto produce significant amounts of heparanase activity. The purificationto homogeneity and characterization of mammalian heparanases has beendifficult, primarily due to the lack of a convenient assay. Most reportscontain only partial description with conflicting information. Oosta, etal. (22) described the purification of a human platelet heparanase withan estimated molecular mass of 134 kDa expressing an endoglucuronidaseactivity. Hoogewert, et al. (23) reported the purification of a 30 kDahuman platelet heparanase which was shown to be an endoglucosaminidasethat cleave both heparin and heparan sulfate essentially todisaccharides. They claimed that the holoenzyme consists of foursubunits, each closely related to the CXC chemokines CTAPIII, NAP-2 andβ-thromboglobulin (23). Freeman and Parish (24) have purified tohomogeneity a 50 kDa platelet heparanase exhibiting endoglucuronidaseactivity. Likewise heparanase enzyme purified from human placenta andfrom hepatoma cell line (U.S. Pat. No. 5,362,641) had a molecular massof approximately 48 kDa. A similar molecular weight was determined bygel filtration analysis of partially purified heparanase enzymesisolated form human platelets, human neutrophils and mouse B16 melanomacells (our unpublished data). In contrast, heparanase purified from B16melanoma cells by Nakajima, et al. (9, 26) had a molecular weight of 96kDa. The latter enzyme has been localized immunochemically to the cellsurface and cytoplasm of human melanoma lesions using a polyclonalantiserum (26) and in tertiary granules in neutrophils using monoclonalantibodies (26a), both directed against a putative amino terminalsequence from purified B16F10 melanoma cell heparanase (26). However,the melanoma heparanase amino terminal sequence was found to becharacteristic of a 94 kDa glucose-regulated protein (GRP94/endoplasmin)that functions as a molecular chaperone which lacks heparanase activity(27). This result and a recent study using anti-endoplasmin antibody(28) suggest that the endoplasmin-like 98 kDa protein found in purifiedmelanoma heparanase preparations is a contaminant (27, 28). This callsinto question the previous heparanase immunolocalization studies carriedout using the B16 melanoma heparanase amino terminal peptide antiserum(26). Likewise, antiserum directed against the amino terminal sequenceof CTAP III was applied to immunolocalize the heparanase enzyme inbiopsy specimens of human prostate and breast carcinomas (29, 30).Again, the validity of the results is questionable, since thepossibility that CTAP III is a contaminant of the platelet preparationwas not excluded. First, attempts to express heparanase activeCTAPIII/NAP2 protein were unsuccessful and the recombinant CTAPIII/NAP2chemokines failed to exhibit heparanase activity. Second, western blotanalysis of the platelet enzyme purified by Freeman and Parish (24) withantibodies against human β-thromboglobulin or platelet factor-4demonstrated that these and related proteins (e.g., CTAP-III and NAP-2)were not present in the purified platelet heparanase preparations (24).Moreover, while heparanase activity can be detected in purifiedpreparations of β-thromboglobulin, it is probably due to contaminationwith the “classical” platelet heparanase since it exhibited anendo-beta-D-glucuronidase activity rather than an endoglucosaminidaseactivity (23), as reported by Hoogewerf et al. (Pikas et al. manuscriptsubmitted for publication).

[0015] Our studies on the immunolocalization of CTAPIII in human biopsyspecimens revealed a preferential localization of CTAP-III in cells(i.e., vascular endothelia cells, keratinocytes) that failed to expressheparanase activity and vice versa. Finally, none of the sequencespublished by Hoogewerf et al (platelet CTAP-III/NAP-2) (23) or Jin etal. (B16 melanoma) (26) nor sequences of the bacterial heparin/heparansulfate degrading enzymes (hep I & III) (30a) were found in ourrecombinant human heparanase that was cloned and expressed on the basisof sequences derived from the purified human placenta and hepatomaheparanases.

[0016] Several years ago we prepared rabbit polyclonal antibodiesdirected against our partially purified preparation of human placentaheparanase. These antibodies, referred to in U.S. Pat. No. 5,362,641,were later found to be directed against plasminogen activator inhibitortype I (PAI-1) that was co-purified with the placental heparanase. Thesefindings led to a modification of the original purification protocol toremove the PAI-1 contaminant.

[0017] Collectively, it is evident that so far no one had succeeded ineliciting anti-heparanase antibodies.

[0018] Unlike the above described information, both the polyclonal andmonoclonal antibodies described hereinunder were raised, for the firsttime, against a purified, highly active, recombinant enzyme. As furthershown below these antibodies specifically recognizes the heparanaseenzyme in cell lysates and conditioned media and does not cross-reactwith β-thromboglobulin, NAP-2, PAI-1 or bacterial heparinases I and III.They do recognize the mouse B16-F10 heparanase, the human plateletheparanases, and the heparanase enzymes produced by several human tumorcell lines and Chinese hamster ovary (CHO) cells. By virtue of beingproduced against a purified recombinant enzyme and their specificity,these antibodies appear highly appropriate for diagnostic purposes suchas immunohistochemistry of biopsy specimens and quantitative ELISA ofbody fluids (e.g., plasma, urine, pleural effusions, etc.). Similarly,as presented in the Examples section hereinunder, both the molecularprobes for in situ determination of the tissue distribution of the hpagene and the cDNA primers for detection of the hpa mRNA in normal andmalignant cells of human origin (e.g., leukemia and lymphoma cells,melanoma cells) can be applied, for the first time, for diagnosis ofearly events in tumor progression, metastatic spread and response totreatment.

SUMMARY OF THE INVENTION

[0019] According to the present invention there are provided heparanasespecific molecular probes and their use in use in research and medicalapplications including diagnosis and therapy.

[0020] According to further features in preferred embodiments of theinvention described below, there is provided an antibody elicited by aheparanase protein or an immunogenical portion thereof, the antibodyspecifically binds heparanase.

[0021] According to still further features in the described preferredembodiments the heparanase protein is recombinant.

[0022] According to still further features in the described preferredembodiments the elicitation is through in vivo or in vitro techniques,the antibody having been prepared by a process comprising the steps of(a) exposing cells capable of producing antibodies to the heparanaseprotein or the immonogenical part thereof and thereby generatingantibody producing cells; (b) fusing the antibody producing cells withmyeloma cells and thereby generating a plurality of hybridoma cells eachproducing monoclonal antibodies; and (c) screening the plurality ofmonoclonal antibodies to identify a monoclonal antibody whichspecifically binds heparanase.

[0023] According to still further features in the described preferredembodiments the antibody is selected from the group consisting of apolyclonal antibody and a monoclonal antibody.

[0024] According to still further features in the described preferredembodiments the polyclonal antibody is selected from the groupconsisting of a crude polyclonal antibody and an affinity purifiedpolyclonal antibody.

[0025] According to further features in preferred embodiments of theinvention described below, there is provided an oligonucleotidecomprising a nucleic acid sequence specifically hybridizable withheparanase encoding nucleic acid.

[0026] According to further features in preferred embodiments of theinvention described below, there is provided a pair of polymerase chainreaction primers comprising a sense primer and an antisense primers,each of the primers including a nucleic acid sequence specificallyhybridizable with heparanase encoding nucleic acid.

[0027] According to further features in preferred embodiments of theinvention described below, there is provided an antisense nucleic acid(RNA or DNA) molecule comprising a nucleic acid sequence specificallyhybridizable with heparanase messenger RNA.

[0028] According to further features in preferred embodiments of theinvention described below, there is provided a sense nucleic acid (RNAor DNA) molecule comprising a nucleic acid sequence specificallyhybridizable with heparanase antisense RNA.

[0029] According to further features in preferred embodiments of theinvention described below, there is provided a method of in situdetecting localization and distribution of heparanase expression in abiological sample comprising the step of reacting the biological samplewith a detectable heparanase specific molecular probe and detecting thelocalization and distribution of the detectable heparanase specificmolecular probe.

[0030] According to further features in preferred embodiments of theinvention described below, there is provided a method of detectingheparanase expression in a biological sample comprising the step ofreacting the biological sample with a detectable heparanase specificmolecular probe and detecting said detectable heparanase specificmolecular probe. Protein and nucleic acid dot blot application areenvisaged.

[0031] According to still further features in the described preferredembodiments the biological sample is selected from the group consistingof cells and tissues.

[0032] According to still further features in the described preferredembodiments the biological sample is malignant.

[0033] According to still further features in the described preferredembodiments the malignancy is selected from the group consisting of asolid tumor and a hematopoietic tumor.

[0034] According to still further features in the described preferredembodiments the solid tumor is selected from the group consisting ofcarcinoma, adenocarcinoma, squameous cell carcinoma, teratocarcinoma,mesothelioma and melanoma, and further wherein the hematopoietic tumoris selected from the group consisting of lymphoma and leukemia.

[0035] According to still further features in the described preferredembodiments the solid tumor is a primary tumor, or a metastasis thereof,and is originated from an organ selected from the group consisting ofliver, prostate, bladder, breast, ovary, cervix, colon, skin, intestine,stomach, uterus, pancreas.

[0036] According to still further features in the described preferredembodiments the detectable heparanase specific molecular probe isselected from the group consisting of a nucleic acid sequencehybridizable with heparanase encoding nucleic acid and ananti-heparanase antibody capable of specifically binding heparanase.

[0037] According to still further features in the described preferredembodiments the nucleic acid sequence hybridizable with heparanaseencoding nucleic acid is selected from the group consisting of asynthetic oligonucleotide, an antisesnse heparanase RNA and heparanaseDNA labeled by a detectable moiety.

[0038] According to further features in preferred embodiments of theinvention described below, there is provided a method of detectingheparanase protein in a body fluid of a patient comprising the steps ofreacting the body fluid with an anti-heparanase antibody and monitoringthe reaction.

[0039] According to still further features in the described preferredembodiments the body fluid is selected from the group consisting ofplasma, urine, pleural effusions and saliva.

[0040] According to still further features in the described preferredembodiments the body fluid is of a patient suffering from a conditionselected from the group consisting of cancer, renal disease anddiabetes.

[0041] According to still further features in the described preferredembodiments the renal disease is associated with diabetes.

[0042] According to still further features in the described preferredembodiments the anti-heparanase antibody is selected from the groupconsisting of a monoclonal antibody and a poly clonal antibody.

[0043] According to still further features in the described preferredembodiments reacting the body fluid with the anti-heparanase antibody iseffected in solution.

[0044] According to still further features in the described preferredembodiments reacting the body fluid with the anti-heparanase antibody iseffected on a substrate capable of adsorbing proteins present in thebody fluid.

[0045] According to still further features in the described preferredembodiments the body fluid is of a patient suffering from myeloma,breast carcinoma, metastatic breast carcinoma, hemorrhagic nephritis,nephrotic syndrome, normoalbuminuric type I diabetes, microalbuminurictype I diabetes, kidney disorder, inflammation, sepsis, inflammatory andautoimmune disease.

[0046] According to further features in preferred embodiments of theinvention described below, there is provided a method of detecting thepresence, absence or level of heparanase transcripts in a biologicalsample comprising the steps of (a) extracting messenger RNA from thebiological sample, thereby obtaining a plurality of messenger RNAs; (b)reverse transcribing the plurality of messenger RNAs into a plurality ofcomplementary DNAs; (c) contacting the plurality of complementary DNAswith a pair of heparanase specific polymerase chain reaction primers,nucleoside triphosphates and a thermostable DNA polymerase; (d)performing a polymerase chain reaction; and (e) detecting the presence,absence or level of the polymerase chain reaction product.

[0047] According to further features in preferred embodiments of theinvention described below, there is provided a method of detectingheparanase messenger RNA in a biological sample comprising the steps ofreverse transcribing the messenger RNA into complementary DNA,contacting the complementary DNA with polymerase chain reactionoligonucleotides hybridizable to heparanase encoding nucleic acid,performing a polymerase chain reaction and monitoring for heparanasespecific polymerase chain reaction products.

[0048] According to further features in preferred embodiments of theinvention described below, there is provided a method of detecting thepresence, absence or level of heparanase protein in a biological samplecomprising the steps of (a) extracting proteins from the biologicalsample, thereby obtaining a plurality of proteins; (b) size separatingthe proteins; (c) interacting the size separated proteins with ananti-heparanase antibody; and (d) detecting the presence, absence orlevel of the interacted anti-heparanase antibody.

[0049] According to still further features in the described preferredembodiments the anti-heparanase antibody is selected from the groupconsisting of a polyclonal antibody and a monoclonal antibody.

[0050] According to still further features in the described preferredembodiments the size separation is effected by electrophoresis.

[0051] According to further features in preferred embodiments of theinvention described below, there is provided a method of targeted drugdelivery to a tissue of a patient, the tissue expressing heparanase, themethod comprising the steps of providing a complex of a drug directly orindirectly linked to an anti-heparanase antibody and administering thecomplex to the patient.

[0052] According to further features in preferred embodiments of theinvention described below, there is provided a method of treating apatient having a condition associated with heparanase expressioncomprising the step of administering an anti-heparanase antibody to thepatient.

[0053] It is an object of the present invention to use a heparanasespecific molecular probe for detection of the presence, absence or levelof heparanase expression.

[0054] It is another object of the present invention to use a heparanasespecific molecular probe for therapy of a condition associated withexpression of heparanase.

[0055] It is yet another object of the present invention to use aheparanase specific molecular probe for quantification of heparanase ina body fluid.

[0056] It is still another object of the present invention to use aheparanase specific molecular probe for targeted drug delivery.

[0057] It is another object of the present invention to use a heparanasespecific molecular probe as a therapeutic agent.

[0058] The present invention successfully addresses the shortcomings ofthe presently known configurations by providing a variety of heparanasespecific molecular probes which can be used for research and medicalapplications including diagnosis and therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The invention herein described, by way of example only, withreference to the accompanying drawings, wherein:

[0060]FIG. 1 demonstrates the expression of the human heparanase gene byhuman breast carcinoma cell lines with different metastatic potentials.Total RNA was isolated and subjected to semi quantitative RT-PCR (28cycles) using human heparanase primers (hep) and primers for the GAPDHhousekeeping gene. Reactions without reverse transcriptase demonstratedno amplification of genomic DNA contamination in the RNA samples (notshown). Lane 1, Non metastatic MCF-7 cells, lane 2, moderate metastaticMDA-231 cells, lane 3, highly aggressive MDA-435 cells, lane 4, minimalmetastatic ZR-75 cells, lane 5, moderate metastatic MCF-ANeoT cells,lane 6, highly metastatic MCF-T₆ 3B cells; lane 7, DNA molecular weightmarker VI (Boehringer Mannheim).

[0061]FIGS. 2a-b demonstrate heparanase activity expressed by humanbreast carcinoma cell lines with different metastatic potentials. Breastcarcinoma cell lysates of the above described cell lines were incubated(24 hours, 37° C., pH 6.2) with ³⁵S-HSPG isolated from intactsubendothelial ECM. Heparanase mediated conversion of the heparansulfate substrate (peak I) into low MW degradation fragments (peak II)was analyzed by gel filtration on SEPHAROSE 6B. Expression of the humanhpa gene correlates with heparanase activity and metastasis inexperimental animals.

[0062]FIGS. 3a-f demonstrate detection of hpa mRNA by in situhybridization in specimens of normal and malignant human breast tissuewith antisense heparanase RNA probe: invasive carcinoma of the breast,pre-malignant fibrocystic breast tissue, adenocarcinoma of the breast,invasive breast carcinoma surrounding the area of tumor necrosis (notstained), normal breast tissue-reduction mammoplasty (antisense hpaprobe), and normal breast tissue-reduction mammoplasty (control senseprobe), respectively.

[0063]FIG. 4 demonstrate heparanase activity expressed by human prostatecarcinoma cell lines. Expression of the human hpa gene by normal andmalignant human prostate cells. Total RNA was isolated and subjected toRT-PCR using the appropriate human hpa primers (hep) and primers for theGAPDH housekeeping gene. Reactions without reverse transcriptasedemonstrated no genomic DNA contamination in the RNA samples (notshown). Lane 1, metastatic DU145 human prostate carcinoma cells, lane 2,metastatic PC3 human prostate carcinoma cells, lane 3, normal humanprostate tissue (biopsy specimen), lane 4, DNA molecular weight markerVI (Boehringer Mannheim).

[0064]FIG. 5 demonstrate the expression of the hpa gene by high and lowmetastatic human bladder carcinoma and mouse T lymphoma cell lines.Total RNA was isolated and subjected to RT-PCR using human hpa primers.Lane 1, non metastatic MBT2 human bladder carcinoma cells, lane 2,highly metastatic T50 variant of MBT2 cells, lane 3, non-metastatic Ebmouse T-lymphoma, lane 4, highly metastatic ESb variant of the Eb mouseT-lymphoma cells, lane 5, DNA molecular weight marker VI (BoehringerMannheim). −RT: negative control, without reverse transcriptase, P: nonamplified primers.

[0065]FIGS. 6a-c demonstrate heparanase activity expressed by high andlow metastatic human bladder carcinoma cells. Media conditioned by low(MBT2) and high (T50) metastatic human bladder carcinoma cells wereincubated (24 hours, 37° C., pH 6.2) with ³⁵S-HSPG isolated from intactsubendothelial ECM. Heparanase mediated conversion of the heparansulfate substrate (peak I, ss 47) into low molecular weight degradationfragments (peak II) was analyzed by gel filtration on SEPHAROSE 6B.Expression of the human hpa gene correlates with heparanase activity andmetastasis in experimental animals.

[0066]FIG. 7 demonstrate expression of the hpa gene by high and lowmetastatic B16 mouse melanoma cell lines. Total RNA was isolated andsubjected to RT-PCR using hpa primers (hep) and primers for the GAPDHhousekeeping gene. Reactions without reverse transcriptase demonstratedno genomic DNA contamination in the RNA samples. Lane 1, highlymetastatic B16-F10 mouse melanoma cells, lane 2, low metastatic B16-F1mouse melanoma cells, lane 3, DNA molecular weight marker VI (BoehringerMannheim).

[0067]FIG. 8a demonstrate expression of the hpa gene by biopsy specimensfrom malignant human melanoma tumors and non-malignant benign nevustissue which were processed for cell culture. Total RNA was isolatedfrom subconfluent cultures and subjected to RT-PCR using human specifichpa primers (hep). Representative cases are shown. Lane 1, malignantmelanoma, lane 2, non-malignant nevus tissue, lane 3, hpa-pcDNA plasmid(positive control), lane 4, negative control (no RNA), lane 5, DNAmolecular weight marker VI (Boehringer Mannheim). Reactions withoutreverse transcriptase (−RT) demonstrated no genomic DNA contamination inthe RNA samples.

[0068]FIG. 8b demonstrates heparanase activity expressed by culturedcells derived from malignant melanoma (patient M-24) and non-malignantnevus tissue (patient M-31). Cultured cells were seeded on sulfatelabeled ECM. Labeled degradation fragments released into the incubationmedium were subjected to gel filtration on SEPHAROSE 6B.

[0069]FIGS. 9a-f demonstrate detection of hpa mRNA by in situhybridization in specimens of human malignant melanoma and normal nevus.FIGS. 9a, c and d—metastatic human melanoma (3 different patients), FIG.9b—non malignant nevus tissue. Labeling is not seen in the nevus tissue,as compared to intense staining of the metastatic melanoma. FIGS. 9e andf—same sections as in Figures c and d stained with hematoxylin-eosine.

[0070]FIGS. 10a-f demonstrate detection of hpa mRNA by in situhybridization in specimens of normal and malignant human liver.Hepatocellular carcinoma (×200), hepatocellular carcinoma (×1000), liveradenocarcinoma, normal adult liver, embryonic liver and control sensestaining of embryonic liver are shown respectively. Labeling is not seenin normal liver cells as compared to intense staining of embryonic andmalignant liver cells.

[0071]FIGS. 11a-f demonstrate detection of hpa mRNA by in situhybridization in specimens of normal and malignant human tissues.Adenocarcinoma of the ovary, normal ovary, squameous cell carcinoma ofthe cervix, normal cervix, colon adenocarcinoma and normal smallintestine are shown respectively.

[0072]FIGS. 12a-f demonstrate detection of hpa mRNA by in situhybridization in specimens of various human tumors. Positive staining ofthe hpa gene was clearly seen in adenocarcinoma of the stomach,teratocarcinoma, well differentiated endometrial adenocarcinoma,adenocarcinoma of the pancreas, mesothelioma, FIGS. 12a-e, respectively.Control, sense staining of human mesothelioma is shown in FIG. 12f.

[0073]FIGS. 13a-b demonstrate expression of heparanase in humanleukemias and lymphomas. Peripheral white blood cells of patients withvarious types of leukemia and lymphoma were isolated and tested forexpression of the human hpa gene. For this purpose, total RNA wasisolated and subjected to RT-PCR using human specific hpa primers.Reactions without reverse transcriptase demonstrated no genomic DNAcontamination in the RNA samples. Peripheral white blood cells ofdifferent patients with chronic lymphocytic leukemia (FIG. 13a, lanes1-5) were isolated and tested for expression of the human hpa gene. 13 aLane 6, hpa-pcDNA plasmid (positive control), lane 7, negative control(no reverse transcriptase), lane 8, DNA molecular weight marker VI(Boehringer Mannheim). Representative patients with various types ofleukemia and lymphoma are shown in FIG. 13b. Lane 1, acute myelocyticleukemia, lane 2, Chronic lymphocytic leukemia (atypical B cell), lane3, acute myelocytic leukemia (M5), lane 4, hairy cell leukemia, lane 5,non-hodjkin lymphoma (mature B cells), lane 6, non-hodjkin lymphoma(mature B cells), lane 7, chronic lymphocytic leukemia (stage I), lane8, acute myelocytic leukemia (M2), lane 9, chronic myelocytic leukemia,lane 10, chronic lymphocytic leukemia (stage II), lane 11, acutelymphocytic leukemia, lane 12, chronic lymphocytic leukemia (stage III),lane 13, acute myelocytic leukemia (M1), lane 14, acute myelocyticleukemia (M3), lane 15, hpa-pcDNA plasmid (positive control), lane 16,negative control (no reverse transcriptase), lane 17, DNA molecularweight marker VI (Boehringer Mannheim).

[0074]FIG. 14 demonstrates no expression of the hpa gene by normal humanumbilical cord white blood cells. Total RNA was isolated and subjectedto RT-PCR using hpa primers (hep) and primers for the GAPDH housekeepinggene. Reactions without reverse transcriptase demonstrated no genomicDNA contamination in the RNA samples. Lanes 1-6, white blood cellpreparations from 6 different umbilical cords, lane 7, hpa-pcDNA plasmid(positive control), lane 8, negative control (no reverse transcriptase),lane 9, DNA molecular weight marker VI (Boehringer Mannheim).

[0075]FIG. 15 demonstrates expression of the hpa gene by leukemia andlymphoma cell lines. Total RNA was isolated and subjected to RT-PCRusing hpa primers (hep) and primers for the GAPDH housekeeping gene.Reactions without reverse transcriptase demonstrated no genomic DNAcontamination in the RNA samples. Lane 1, normal B lymphoblastoid cellline (Monga), lane 2, Burkitt B lymphoma (Raji), lane 3, Burkitt Blymphoblasts (Daudi), lane 4, Burkitt B lymphoblasts (non Ebv, DG-75),lane 5, erythroleukemia (K-562), lane 6, pre B lymphoma (nalm₆), M=DNAmolecular weight marker VI (Boehringer Mannheim).

[0076]FIGS. 16a-h demonstrate urinary heparanase activity. Urine samples(o) of healthy donor (16 d) and patients with multiple myeloma (16 a),bilateral breast carcinoma (16 b), metastatic breast carcinoma (16 c),hemorrhagic nephritis (16 e) nephrotic syndrome (16 f), normoalbuminuric(16 g) and microalbuminuric type I diabetes (16 h) were incubated (24hours, 37° C., pH 6.2) with ³⁵S-HSPG (50 μl) isolated from intactsubendothelial ECM (♦). Heparanase mediated conversion of the heparansulfate substrate (peak I) into low molecular weight degradationfragments (peak II) was analyzed by gel filtration on SEPHAROSE 6B.

[0077]FIGS. 17a-b demonstrate Western blots of extracts of cellsexpressing various segments of heparanase as detected with polyclonalanti heparanase antibodies. 17 a—antiserum from rabbit 7640, 17b—antiserum from rabbit 7644. Lane 1, E. coli BL21(DE3)pLysS cellstransfected with pRSET, lane 2, E. coli BL21(DE3)pLysS cells transfectedwith pRSET containing the heparanase entire open reading frame (543amino acids, SE ID NOs: 2 and 3), lane 3, E. coli BL21(DE3)pLysS cellstransfected with pRSEThpaBK containing 414 amino acids of the heparanaseopen reading frame (amino acids 130-543 of SEQ ID NOs: 2 and 3), lane 4,E. coli BL21(DE3)pLysS cells transfected with pRSEThpaBH containing 302amino acids of the heparanase open reading frame (amino acids 130-431 ofSEQ ID NOs: 2 and 3), lane 5, molecular size markers, lane 6, medium ofSf21 insect cells infected with recombinant Baculovirus pFhpa containingthe heparanase entire open reading frame (543 amino acids, SEQ ID NOs: 2and 3), lane 7, Sf21 insect cells infected with recombinant baculoviruswith no insert. Proteins were separated on 10% SDS-PAGE, antisera werediluted 1:1,000. Detection was performed by ECL (Amersham) according tothe manufacturer's instructions. Size in kDa is shown to the right, aswas determined using prestained SDS-PAGE standards, Bio-Rad, CA.

[0078]FIG. 18 demonstrates Western blot using affinity purifiedpolyclonal antibodies with heparanase expressed in various expressionsystems. Lane 1, medium of Sf21 insect cells infected with recombinantBaculovirus pFhpa, lane 2, cell extract of a Chinese hamster ovary (CHO)clone stably transfected with a vector containing no insert, lane 3,cell extract of a CHO stable clone transfected with hpa cDNA, lane 4,proteins precipitated from medium of the yeast Pichia pastoristransfected with hpa cDNA. Proteins were separated on 4-20% gradientSDS-PAGE, antibody was diluted 1:100. Detection was performed by ECL(Amersham) according to the manufacturer's instructions. For CHO andPichia clones see U.S. patent application Ser. No. 09/071,618, which isincorporated by reference as if fully set forth herein. Size in kDa isshown to the right, as was determined using prestained SDS-PAGEstandards, Bio-Rad, CA.

[0079]FIGS. 19a-b demonstrate Western blot of extracts of various celltypes using anti-heparanase polyclonal antibodies. 19 a—crude antiserumdiluted 1:2,000, 19 b—affinity purified antibodies diluted 1:100. lane1, purified heparanase from placenta, lanes 2 and 3, cell extracts ofplatelets, insoluble and soluble fractions, respectively, lanes 4 and 5,cell extracts of neutrophils, insoluble and soluble fractions,respectively, lanes 6 and 7, cell extracts of mouse melanoma B16-F1cells, insoluble and soluble fractions, respectively. Proteins wereseparated on 8-16% gradient gel. Detection was performed by ECL(Amersham) according to the manufacturer's instructions. Size in kDa isshown to the right, as was determined using prestained SDS-PAGEstandards, Bio-Rad, CA.

[0080]FIG. 20 demonstrates Western blot of recombinant and nativeheparanases from various origins using supernatant of hybridoma HP-117.Lanes 1 and 2, 293 human kidney cells non-transfected and transfectedwith hpa-pCDNA, respectively (15 μg), lane 3, CHO cells stablytransfected with pShpa (40 μg), lane 4, mock transfected CHO cells (40μg), lane 5, purified recombinant heparanase produced by baculovirusinfected insect cells (50 ng), lane 6, cell extracts of E. coliexpressing recombinant heparanase (50 ng), lane 7, cell extract of humanplatelets (100 μg), lane 8, prestained SDS-PAGE standard, Bio-Rad, CA.Proteins were separated on 4-20% gradient SDS-PAGE and transferred to anylon membrane (Amersham). Membrane was incubated with supernatant ofhybridoma Hp117 and detection was performed with alkaline phosphataseconjugated anti-mouse IgG antibodies.

[0081]FIGS. 21a-b demonstrate immunostaining of heparanase in CHO cellswith polyclonal antibodies. CHO cells transfected with the full lengthhpa gene (21 a) were tested for overexpression of heparanase. Stainingis detected in the cytoplasm of transfected cells. In non transfectedCHO cells (21 b), no staining of heparanase is detected.

[0082]FIGS. 22a-b demonstrate immunostaining of heparanase in CHO cellswith monoclonal antibody HP-130. CHO cells transfected with the fulllength hpa gene (22 a) were tested for overexpression of heparanase.Staining is detected in the cytoplasm of transfected cells. In nontransfected CHO cells (22 b), no staining of heparanase is detected.

[0083]FIGS. 23a-c demonstrate immunostaining of heparanase in bloodsmears from normal donor with monoclonal antibody HP-92. Heparanase isfound in the cytoplasm of neutrophils (23 a) and platelets (23 c) but isnot detected in lymphocytes (23 b) and monocytes (23 c).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0084] The present invention is of heparanase specific molecular probeswhich can be used in research and medical applications. Specifically,the present invention can be used for the detection and monitoring ofmalignancies, metastasis and other, non-malignant conditions, efficiencyof therapeutic treatments, targeted drug delivery and therapy, usingheparanase specific molecular probes, such as anti-heparanase antibodies(both poly- and monoclonal) and heparanase gene (hpa) derived nucleicacids, including, but not limited to, PCR primers, antisenseoligonucleotide probes, antisense RNA probes, DNA probes and the like.

[0085] The principles and operation of the present invention may bebetter understood with reference to the drawings and accompanyingdescriptions.

[0086] Before explaining at least one embodiment of the invention indetail, it is to be understood that the invention is not limited in itsapplication to the details of construction and the arrangement of thecomponents set forth in the following description or illustrated in thedrawings. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

[0087] As shown in the Examples section below heparanase specificantibodies and/or nucleic acids reveals in situ expression (proteinand/or messenger RNA) of heparanase in a variety of cells and tissues,especially in malignant cells and tissues, wherein the degree ofexpression corroborates with metastasis.

[0088] Therefore, according to one aspect of the present invention thereis provided a method of in situ detecting localization and distributionof heparanase expression in a biological sample. The method comprisesthe step of reacting the biological sample with a detectable heparanasespecific molecular probe and detecting the localization and distributionof the detectable heparanase specific molecular probe.

[0089] According to another aspect of the present invention, there isprovided a method of detecting heparanase expression in a biologicalsample. The method comprises the step of reacting the biological samplewith a detectable heparanase specific molecular probe and detecting thedetectable heparanase specific molecular probe. Protein and nucleic aciddot blot application are envisaged.

[0090] As used herein in the specification and in the claims sectionbelow, the term “heparanase expression” refers mainly to the processesof transcription and translation, resulting in a catalytically activeheparanase having endoglycosidase hydrolyzing activity which is specificfor heparin or heparan sulfate proteoglycan substrates, as opposed tothe activity of bacterial enzymes (heparinase I, II and III) whichdegrade heparin or heparan sulfate by means of β-elimination.

[0091] As used herein in the specification and in the claims sectionbelow, the term “biological sample” refers to cells and tissues,including, but not limited to cancer cells and tissues. The term furtherrelates to body fluids, as further detailed below.

[0092] As used herein in the specification and in the claims sectionbelow, the term “detectable heparanase specific molecular probe” and itsequivalent term “detectable heparanase molecular probe” both refer to anucleic acid sequences hybridizable with heparanase encoding nucleicacid or to an anti-heparanase antibody capable of specifically bindingheparanase. The nucleic acid sequence hybridizable with heparanaseencoding nucleic acid is, for example, a synthetic oligonucleotide, anantisesnse heparanase RNA or heparanase DNA, and it is preferablylabeled by the detectable moiety.

[0093] As used herein in the specification and in the claims sectionbelow, the term “detectable moiety” refers to any atom, molecule or aportion thereof, the presence, absence or level of which is directly orindirectly monitorable. One example include radioactive isotopes. Otherexamples include (i) enzymes which can catalyze color or light emitting(luminescence) reactions and (ii) fluorophores. The detection of thedetectable moiety can be direct provided that the detectable moiety isitself detectable, such as, for example, in the case of fluorophores.Alternatively, the detection of the detectable moiety can be indirect.In the latter case, a second moiety reactable with the detectablemoiety, itself being directly detectable is preferably employed. Thedetectable moiety may be inherent to the molecular probe. For example,the constant region of an antibody can serve as an indirect detectablemoiety to which a second antibody having a direct detectable moiety canspecifically bind.

[0094] As used herein in the specification and in the claims sectionbelow, the term “antibody” refers to any monoclonal or polyclonalimmunoglobulin, or a fragment of an immunoglobin such as sFv (singlechain antigen binding protein), Fab1 or Fab2. The immunoglobulin couldalso be a “humanized” antibody, in which murine variable regions arefused to human constant regions, or in which murinecomplementarity-determining regions are grafted onto a human antibodystructure (Wilder, R. B. et al., J. Clin. Oncol., 14:1383-1400, 1996).Unlike mouse or rabbit antibodies, “humanized” antibodies often do notundergo an undesirable reaction with the immune system of the subject.The terms “sFv” and “single chain antigen binding protein” refer to atype of a fragment of an immunoglobulin, an example of which is sFv CC49(Larson, S. M. et al., Cancer, 80:2458-68, 1997).

[0095] According to one embodiment of the invention the biologicalsample is malignant, e.g., it is a solid tumor or hematopoietic tumorsample. The solid tumor can, for example, be of the types: carcinoma,adenocarcinoma, squameous cell carcinoma, teratocarcinoma, mesotheliomaor melanoma, which are shown hereinunder in the Examples section toexpress heparanase in good correlation to the degree of metastasis. Thehematopoietic tumor can, for example, be lymphoma or leukemia.

[0096] In some embodiments of the present invention the solid tumor is aprimary tumor, or a metastasis thereof, and it originates from an organsuch as, for example, liver, prostate, bladder, breast, ovary, cervix,colon, skin, intestine, stomach, uterus (including embryo) and pancreas.

[0097] As shown in the Examples section below, it was further found thatbody fluids (e.g., urine) of patients with certain conditions includecatalitically active heparanase. These conditions include myeloma,breast carcinoma, metastatic breast carcinoma, hemorrhagic nephritis,nephrotic syndrome, normoalbuminuric type I diabetes, microalbuminurictype I diabetes, kidney disorder, inflammation, sepsis, inflammatory andautoimmune disease.

[0098] Therefore, according to another aspect of the present inventionthere is provided a method of detecting heparanase protein in a bodyfluid of a patient. The method comprises the steps of reacting the bodyfluid with an anti-heparanase antibody, either poly or monoclonalantibody, and monitoring the reaction. The body fluid is, for example,plasma, urine, pleural effusions or saliva. Monitoring the reaction maybe effected by having the antibody labeled with a detectable moiety, orto use its constant region as an inherent detectable moiety, to which asecond antibody which includes a detectable moiety can specificallybind.

[0099] Urine heparanase was detected in patients suffering fromconditions such as cancer, renal disease and diabetes. In some cases therenal disease was associated with diabetes.

[0100] According to a preferred embodiment of the present inventionreacting the body fluid with the anti-heparanase antibody is effected insolution. Alternatively, reacting the body fluid with theanti-heparanase antibody is effected on a substrate capable of adsorbingproteins present in the body fluid, all as well known in the art ofantibody based diagnosis.

[0101] As further shown in the Examples section below, RT-PCR provesuseful in detecting the presence, absence or level of heparanasetranscripts in various biological samples.

[0102] Therefore, according to another aspect of the present inventionthere is provided a method of detecting the presence, absence or levelof heparanase transcripts in a biological sample. The method comprisesthe following steps. First, messenger RNA (e.g., as a component of totalRNA) is extracted from the biological sample, thereby a plurality ofmessenger RNAs are obtained. Second, the plurality of messenger RNAs arereverse transcribed into a plurality of complementary DNAs. Third, theplurality of complementary DNAs are contacted with a pair of heparanasespecific polymerase chain reaction (PCR) primers, nucleosidetriphosphates and a thermostable DNA polymerase (e.g., Thermophilusaquaticus DNA polymerase, native or recombinant) and a polymerase chainreaction is performed by temperature cycling, as well known in the art.Finally, the presence, absence or level of the polymerase chain reactionproduct is detected, e.g., by gel electrophoresis, by monitoring theincorporation of a detectable moiety into the product or any otherapplicable way, all as well known in the art.

[0103] As further shown in the Examples section below, protein blots andanti-heparanase antibodies prove useful in detecting the presence,absence or level of heparanase protein in various biological samples.

[0104] Therefore, further according to the present invention there isprovided a method of detecting the presence, absence or level ofheparanase protein in a biological sample. The method comprises thefollowing steps. First, proteins are extracted from the biologicalsample, thereby a plurality of proteins are obtained. The proteinextract may be a crude extract and can also include non-proteinaciousmaterial. Second, the proteins are size separated, e.g., byelectrophoresis, gel filtration etc. Fourth, the size separated proteinsare interacted with an anti-heparanase antibody, either poly ormonoclonal antibody. Finally, the presence, absence or level of theinteracted anti-heparanase antibody is detected. In case of gelelectrophoresis the interaction with the antibody is typically performedfollowing blotting of the size separated proteins onto a solid support(membrane).

[0105] In many cases it was shown that directly or indirectly (e.g., vialiposomes) linking a drug (e.g., anti cancerous drug, such as, forexample radio isotopes) to an antibody which recognized a proteinspecifically expressed by a tissue sensitive to the drug andadministering the antibody-drug complex to a patient, results intargeted delivery of the drug to the expressing tissue.

[0106] Therefore, according to yet another aspect of the presentinvention there is provided a method of targeted drug delivery to atissue of a patient, the tissue expressing heparanase. The methodcomprises the steps of providing a complex of a drug directly orindirectly linked to an anti-heparanase antibody and administering thecomplex to the patient. External radio imaging is also envisaged,wherein the drug is replaced with an imageable radio isotope. Endoscopicor laparoscopic imaging is also envisaged. In the latter cases the drugis typically replaced by a fluorescence or luminescence substance. Theseprocedures may, for example, be effective in finding/destroyingmicrometastases.

[0107] In other cases, it was shown that administering an antibodycapable of binding epitopes associated with certain tissues providemeans of destroying such tissues by an elicited immune response.

[0108] Therefore, according to another aspect of the present inventionthere is provided a method of treating a patient having a conditionassociated with heparanase expression. The method comprises the step ofadministering an anti-heparanase antibody to the patient.

[0109] Further according to the present invention there is provided anantibody elicited by a heparanase protein (e.g., recombinant) or animmunogenical portion thereof, the antibody specifically bindsheparanase. The antibody can be a poly or monoclonal antibody. If it ispoly clonal and produced in vivo, it is preferably affinity purified,however crude antibody preparations are also applicable, all as shownand described in more detail in the Examples section hereinunder.

[0110] Preferably, the elicitation of the antibody is through in vivo orin vitro techniques, the antibody having been prepared by a processcomprising the steps of, first, exposing cells capable of producingantibodies to the heparanase protein or the immonogenical part thereofand thereby generating antibody producing cells. Second, fusing theantibody producing cells with myeloma cells and thereby generating aplurality of hybridoma cells each producing monoclonal antibodies, andthird, screening the plurality of monoclonal antibodies to identify amonoclonal antibody which specifically binds heparanase.

[0111] Further according to the present invention there is provided anoligonucleotide comprising a nucleic acid sequence specificallyhybridizable with heparanase encoding nucleic acid, be it heparanase DNAor RNA. The oligonucleotide may include natural nucleotides and/ornucleotide analogs, such as, but not limited to phosphorothioatedanalogs. Such oligonucleotides are readily synthesized provided that thesequence is known. Such oligonucleotides can be deduces, for example,from SEQ ID NOs: 1 and 3.

[0112] Further according to the present invention there are provided anantisense nucleic acid (RNA or DNA) molecule comprising a nucleic acidsequence specifically hybridizable with heparanase messenger RNA and asense nucleic acid (RNA or DNA) molecule comprising a nucleic acidsequence specifically hybridizable with heparanase antisense RNA.

EXAMPLES

[0113] Reference is now made to the following examples, which togetherwith the above descriptions, illustrate the invention in a non limitingfashion.

[0114] Experimental Methods and Materials

[0115] Cells: Cultures of bovine corneal endothelial cells (BCECs) wereestablished from steer eyes as previously described (19, 31). Stockcultures were maintained in DMEM (1 gram glucose/liter) supplementedwith 10% newborn calf serum, 5% fetal calf serum (FCS). bFGF (1 ng/ml)was added every other day during the phase of active cell growth (14,15).

[0116] Preparation of sulfate labeled substrates: BCECs (second to fifthpassage) were plated into 35 mm tissue culture plates at an initialdensity of 2×10⁵ cells/ml and cultured in DMEM supplemented with 10% FCSand 5% dextran T-40 for 12 days. Na₂ ³⁵SO₄ (25 μCi/ml) was added on day1 and 5 after seeding and the cultures were incubated with the labelwithout medium change. The subendothelial ECM was exposed by dissolving(5 min, room temperature) the cell layer with PBS containing 0.5% TritonX-100 and 20 mM NH₄OH, followed by four washes with PBS. The ECMremained intact, free of cellular debris and firmly attached to theentire area of the tissue culture dish (14, 15, 20).

[0117] To prepare soluble sulfate labeled proteoglycans (peak Imaterial), the ECM was digested with trypsin (25 μg/ml, 6 hours, 37°C.), the digest was concentrated by reverse dialysis, applied onto aSEPHAROSE 6B gel filtration column and the high molecular weightmaterial (Kav<0.2, peak I) was collected (32). More than 80% of thelabeled material was shown to be composed of heparan sulfateproteoglycans (11).

[0118] Heparanase activity: Cells (1×10⁶/35-mm dish), cell lysates orconditioned medium were incubated on top of ³⁵S-labeled ECM (18 hours,37° C.) in the presence of 20 mM phosphate or phosphate citrate buffer(pH 6.2). Cell lysates and conditioned media were also incubated withsulfate labeled peak I material (10-20 μl). The incubation medium wascollected, centrifuged (18,000 g, 4° C., 3 min), and sulfate labeledmaterial was analyzed by gel filtration on a SEPHAROSE CL-6B column(0.9×30 cm). Fractions (0.2 ml) were eluted with PBS at a flow rate of 5ml/hour and counted for radioactivity using Bio-fluor scintillationfluid. The excluded volume (V_(o)) was marked by blue dextran and thetotal included volume (V_(t)) by phenol red. The latter was shown tocomigrate with free sulfate (11, 20). Degradation fragments of HS sidechains were eluted from SEPHAROSE 6B at 0.5<Kav<0.8 (peak II) (11, 20).A nearly intact HSPG released from ECM by trypsin was eluted next toV_(o) (Kav<0.2, peak I). Recoveries of labeled material applied on thecolumns ranged from 85 to 95% in different experiments.

[0119] Construction of heparanase expression vector: A BamHI-KpnI 1.3 kbfragment (nucleotides 450-1721 of the hpa sequence, SEQ ID NOs: 1 and 3,U.S. Pat. No. 5,968,822) was cut out from pfasthpa and cloned intopRSET-C bacterial expression vector (Invitrogen). The resultingrecombinant plasmid pRSEThpaBK encodes a fusion protein comprised of Histag, a linker sequence and amino acids 130-543 of the heparanase protein(SEQ ID NOs: 2 and 3).

[0120] A 1.6 kb fragment of hpa cDNA was amplified from pfasthpa (a hpacDNA cloned in pfastBac, see U.S. Pat. No. 5,968,822), by PCR usingspecific sense primer: (Hpu-550Nde)—5′-CGCATATGCAGGACGTCGTG GACCTG-3′(SEQ ID NO:4) and a vector specific antisense primer: (3′pFast)5′-TATGATCCTCTAGTACTTCTCGAC-3′ (SEQ ID NO:5). The upper primerintroduced an NdeI site and an ATG codon preceding nucleotide 168 ofhpa. The PCR product was digested by NdeI and BamHI and its sequence wasconfirmed. pRSEThpaBK was digested with NdeI and BamHI and ligated withthe NdeI-BamHI hpa fragment. The resulting plasmid, designatedpRSEThpaS1, encoded an open reading frame of 508 amino acids (36-543) ofthe heparanase protein, lacking the N-terminal 35 amino acids which arepredicted to be a signal peptide. Expression constructs were introducedinto E. coli BL21 (DEL3)pLysS cells (Stratagene), according tosupplier's protocol.

[0121] Preparation of antigen: E. coli cells harboring the recombinantplasmid were grown at 37° C. overnight in Luria broth containingampicillin and chloramphenicol. Cells were diluted 1/10 in the samemedium, and the cultures were grown to an OD600 of approximately 0.5.Isopropyl-thiogalactoside (IPTG) (Promega) was added to a finalconcentration of 1 mM and the culture was incubated at 37° C. for 3hours. Cells from induced cultures were cooled on ice, sedimented bycentrifugation at 4,000×g for 20 minutes at 4° C., and resuspended in0.5 ml of cold phosphate-buffered saline (PBS). Cells were lysed bysonication, and cell debris was sedimented by centrifugation at 10,000×gfor 20 minutes. The resulting pellet was analyzed by 10% SDS-PAGE. Thegel was stained with 1×PBS coomassie blue and the band of 45 kDa whichcontained the recombinant heparanase was cut out and crashed through aneedle (21G) attached to a syringe. For immunization of mice, thecrashed gel was incubated in PBS overnight at 4° C. and the proteindiffused into the buffer was collected. Rabbits ware injected with gelhomogenate.

[0122] The 55 kDa protein (508 amino acids) was purified from E. coliinclusion bodies by preparative SDS-PAGE, using a Model 491 Prep Cell(Bio-Rad) which is designed to purify proteins from complex mixtures bycontinuous elution electrophoresis. This antigen was used for ELISAscreening.

[0123] Immunization—polyclonal antibodies: Two rabbits (designated 7640and 7644) were immunized each with 200 μg of protein emulsified withequal volume of complete Freund's adjuvant. An equal amount of proteinemulsified with incomplete Freund's was injected to each rabbit twoweeks following the first injection and again after another four weeks.Ten days after the third injection the rabbits were bled and serum wasexamined for reactivity with recombinant heparanase. Four weeks afterbleeding another boost was injected and 10 days later blood wascollected.

[0124] Immunization—monoclonal antibodies: 6 to 8 weeks old femaleBalb/C mice were each immunized intradermally with 50 μg recombinantheparanase emulsified in 50 μl PBS complete Freund's adjuvant. Two tothree weeks later the same amount of the emulsion was injectedsubcutaneously or intradermally at multiple sites in incomplete Freund'sadjuvant. After 3 weeks 25 μg antigen in aqueous solution was injectedintrapertonealy. 7-10 days later animals were bled and the titer of therelevant antibodies was determined. 3-4 weeks after the last boost, oneor two animals were injected intraperitoneal with 20 μg of solubleantigen (in PBS) and 3-4 days later spleens were removed.

[0125] Fusion and cloning: The spleens of immunized mice were ground,splenocytes were harvested and fused with the NSO myeloma cells byadding 41% PEG. Hybridoma cells were grown in HAT-selective DMEM growthmedia containing 15% (v/v) HS (Beit Haemek), 2 mM glutamine,Pen-Strep-Nystatin solution (Penicillin: 10,000 units/ml, Streptomycin:10 mg/ml, Nystatin: 1,250 units/ml), at 37° C. in 8% CO₂ containingatmosphere. Hybridoma cells were cloned by limiting dilution. Hybridomasproducing Mabs to human heparanase were identified by reactivity withsolid-phase immobilized human heparanase.

[0126] ELISA: Falcon polyvinyl plates were coated with 50 ng/well ofbaculovirus derived human heparanase (native) and 100 ng/well of E. coliderived human heparanase (55 kDa—non-active) in PBS (pH 7.2) overnightat 40° C. Hybridoma tissue culture supernatants were added to the wells,and incubated at room temperature for 2 hours. Binding of Mabs was thendetected by incubation with HRP-conjugated goat anti mouse IgG (Fabspecific) (Sigma), followed by development in o-phenylenediaminesubstrate (Sigma) and measurement of absorbencies at 450 nm. PBS with0.05% Tween was used to wash the plates between incubations. Polyclonalrabbit anti human heparanase was used as positive control and negativecontrol included coating with PBS or irrelevant supernatant.

[0127] Affinity purification of polyclonal antibodies: 200 μg ofrecombinant heparanase were separated on 10% SDS-PAGE. Followingelectrophoresis protein was transferred to a nitrocellulose membrane(Schleicher & Scuell). Membrane was stained with Ponceau S and theheparanase band was cut out. The membrane strip was blocked for 2 hoursin TBS containing 0.02% Tween 20 and 5% skim milk. Antiserum was diluted1:3 in blocking solution and incubated with the membrane for 16 hours.Membrane strip was washed with 0.15 M NaCl for 20 minutes and then withPBS for additional 20 minutes. Antibodies were eluted with 0.2 Mglycine, 1 mM EDTA pH 2.8 for 20 minutes at room temperature, and thenneutralized by addition of 0.1 volumes of 1 M Tris pH 8.0 and 0.1volumes of 10×PBS. NaNO₃ was added to a final concentration of 0.02%.

[0128] Western blot: Proteins were separated on 4-20%, or 8-16%polyacrylamide ready gradient gels (Novex). Following electrophoresisproteins were transferred to Hybond-P nylon membrane (Amersham) (350mA/100V for 90 minutes). Membranes were blocked in TBS containing 0.02%Tween 20 and 5% skim milk for 1-16 hours, and then incubated withantisera diluted in blocking solution. Blots were then washed inTBS-Tween, incubated with appropriate HRP-conjugated anti mouse/antirabbit IgG, and developed using ECL reagents (Amersham) according to themanufacturer's instructions. Alternatively, an alkaline phosphataseconjugated anti-mouse/anti-rabbit IgG antibodies were used as secondaryantibodies and blots were developed with FAST™ BCIP/NBT (Sigma)according to the supplier's instructions.

[0129] Expression of the heparanase gene in various cell types andtissues (RT-PCR): RT-PCR was applied to evaluate the expression of thehpa gene by various cell types. For this purpose, total RNA was reversetranscribed and amplified, using the following cDNA primers: Humanhpa-Hpu-355 5′-TTCGATCCCAAGAAGGAATCAAC-3′ and (SEQ ID NO:6) Hpl-2295′-GTAGTGATGCCATGTAACTGAATC-3′. (SEQ ID NO:7)

[0130] Expression pattern of the heparanase gene transcript (in situhybridization). In situ hybridization enables determination of thedistribution of hpa transcripts in normal and malignant tissues. Forthis purpose, thin sections of biopsy specimens were processed for insitu hybridization and hybridized with an antisense RNA probe to the hpagene. The experiments have the resolution power to unambiguouslyidentify the expressing cell type, be they tumor cells, tissuemacrophages, mast cells or platelets. Sections were treated withproteinase K to expose the target RNA and to block non specific bindingsites before addition of the probe (34). For in situ hybridization, twodigoxigenin labeled probes were prepared, one in the sense direction andthe other in the anti-sense direction. They were both transcribed from afragment of about 624 bp of the hpa cDNA sequence (nucleotides 728-1351,SEQ ID NOs: 1 and 3) cloned in to the EcoRI-HindIII sites of thetranscription vector pT3T7-Pac (a modified vector derived from pT3T7,Pharmacia), using T3 (for antisense) or T7 (for sense) RNA polymerase,according to the suppliers protocol. Slides were hybridized underappropriate conditions with the labeled probe and the hybridized probeis visualized using colorimetric reagents (NBT & BCIP) (34). Reactionswere stopped when the desired intensity has been reached.

[0131] In situ detection of heparanase by antibodies: hpa-transfectedand non transfected CHO cells were plated on 8-chamber tissue cultureslides (Nunc). Cells were fixed in 95% ethanol, 5% acetic acid for 5minutes at −20° C. Cells were permeabilized using permeabilizationbuffer (20 mM HEPES, pH 7.4; 300 mM Sucrose; 50 mM NaCl; 3 mM MgCl₂;0.5% Triton X-100) for 4 minutes at 4° C. Endogenous peroxidases wereblocked using 0.3% H₂O₂ in methanol and non specific binding sites wereblocked using 5% horse serum in PBS. Monoclonal anti-heparanase antibody(supernatant of hybridoma) was applied and incubated with the cellsovernight at room temperature. Antibody was washed away and biotinylatedsecondary antibody (horse-anti mouse, Vector, Vectastain ABC system) wasadded for 30 minutes at room temperature. Immunostaining was detectedusing Di Amino Benzidine and H₂O₂ (Sigma tablets) until desiredstaining-intensity was achieved. Slides were counterstained with Mayer'shematoxylin. Immunostaining with polyclonal antibodies was performedunder the same conditions, affinity purified antibody was used at 1:500dilution. Biotinylated horse anti-rabbit was used as a secondaryantibody (Vector, Vectastain ABC system). Blood smears were preparedfrom a healthy donor. Fixation and staining were performed as describedabove.

[0132] Experimental Results

[0133] Differential expression of the hpa gene in human breast carcinomaand breast carcinoma cell lines: Semi-quantitative RT-PCR was applied toevaluate the expression of the hpa gene by human breast carcinoma celllines exhibiting different degrees of metastasis (35, 36). While thenon-metastatic MCF-7 breast carcinoma line failed to express theexpected 585 bp cDNA of the hpa gene (FIG. 1, lane 1), moderate (MDA231, FIG. 1, lane 2) and highly (MDA 435, lane 3) metastatic breastcarcinoma cell lines exhibited a marked increase in hpa gene expression.The differential expression of the hpa gene was reflected by a similardifferential pattern of heparanase activity. As demonstrated in FIG. 2a,lysates of MCF-7 cells exhibited little or no heparanase activity, ascompared to a moderate and high activity expressed by MDA-231 andMDA-435 cells, characterized by moderate and high metastatic potentialin nude mice, respectively.

[0134] The same pattern of hpa gene expression and heparan sulfatedegrading activity was observed in another model of breast cancer. Whilethe ZR75 (=MCF10A) displastic breast cell line originated fromfibrocystic breast epithelial cells showed little or no expression ofthe hpa gene (FIG. 1, lane 4), Ha-ras transfected ZR75 cell line(MCF10AT and MCF10AT3B) expressed the hpa gene (lanes 5 and 6) incorrelation with their metastatic potential. The highly metastaticMCF10AT3B cells were derived from the third generation of xenograftedtumors (36). The heparanase activity expressed by these cell lines wasin correlation with their metastatic behavior (FIG. 2b).

[0135] In subsequent experiments, sense and antisense deoxigenin labeledRNA probes (600 bp fragment of the hpa cDNA) were employed to screenarchivial paraffin embedded human breast tissue for expression of thehpa gene transcripts by in situ hybridization.

[0136] As shown in FIGS. 3a-f, massive expression of the hpa gene wasobserved in invasive breast carcinoma (3 a) and breast adenocarcinoma (3c). The hpa gene was already expressed by differentiated epithelialcells of pre-malignant fibrocystic breast (3 b) and in breast carcinomatissue surrounding the area of tumor necrosis where little or nostaining was observed (3 d). Unlike the malignant tissue, normal breasttissue failed to express the hpa transcript as revealed by the lack ofstaining in tissue derived from reduction mammoplasty, both by theantisense (3 e) and sense (3 f) hpa probes.

[0137] Altogether, these results demonstrate a preferential expressionof the hpa gene malignant breast carcinoma cells, indicating a potentialapplication in early diagnosis of the disease, particularly in view ofthe positive staining detected already in the fibrocystic stage.

[0138] Human prostate and bladder carcinomas: Differential expression ofthe hpa mRNA was also suggested by RT-PCR analysis of several humanprostate and bladder carcinoma cell lines. As demonstrated in FIG. 4,both DU145 (lane 1) and PC3 (lane 2) human prostate cell lines showedhigh expression of the hpa mRNA in contrast to lack of, ornon-detectable, expression in a biopsy of normal adult prostate tissue(lane 3). Similarly, as demonstrated in FIG. 5, highly metastaticvariant (T50) of the non-metastatic MBT2 human bladder carcinoma cellline, exhibited a much higher expression of the hpa gene (lane 2) ascompared with the MBT2 cell line (lane 1). This difference was alsoreflected by high heparanase activity secreted into the culture mediumof the aggressive T50 cells, as compared to no detectable activity inthe medium of the parental MBT2 cells (FIGS. 6a-c). Again, the observeddifferential expression of the hpa gene and enzyme activity pointstoward potential application in the diagnosis of metastatic humanprostate and bladder carcinomas.

[0139] Mouse melanoma and T-lymphoma: Differential expression of the hpamRNA and heparan sulfate degrading activity, correlated with themetastatic potential in mice was also demonstrated in studies with mouseB16 melanoma and T-lymphoma. In fact, the melanoma (9, 37) and lymphoma(11) cell systems were the first experimental systems pointing toward animportant role of heparanase in tumor cell invasion and metastasis. Ourcloning of the hpa cDNA, encoding for the heparanase enzyme, provides,for the first time, an evidence that the difference in enzymaticactivity is due primarily to a preferential expression of the hpa geneby highly metastatic tumor cells. Thus, as demonstrated in FIGS. 5 and7, the highly metastatic ESb lymphoma (FIG. 5, lane 4) and B16-F10melanoma (FIG. 7, lane 1) cell lines, expressed the hpa gene to a muchhigher extent as compared to the parental low metastatic Eb lymphoma(FIG. 5, lane 3) and B16-F1 melanoma (FIG. 7, lane 2) cells. Therespective high and low levels of heparanase activity by these celllines were reported in earlier studies (9, 11, 37).

[0140] Human melanoma: Preferential expression of the hpa gene andenzyme activity was also observed in cells derived from biopsies ofhuman melanoma and normal nevus tissue. Biopsy specimens of malignantmelanoma are routinely processed for cell culture in the department ofOncology (Hadassah Hospital, Jerusalem) for immunotherapy purposes.Cultured cells derived from 16 out of 16 patients (see also Table 1,below) expressed the hpa gene, as revealed by RT-PCR (FIG. 8a, lane 1, arepresentative patient). Melanoma cells derived from 3 of these patientswere tested for degradation of soluble heparan sulfate proteoglycans andwere found to be highly active (FIG. 8b). In contrast, cells derivedfrom a non-malignant nevus tissue showed no detectable expression of thehpa mRNA (FIG. 8a, lane 2) and no enzyme activity (FIG. 8b).

[0141] Similar results were obtained using archivial paraffin embeddedbiopsy specimens and in situ hybridization. Again, cytoplasmic labelingof the hpa mRNA was observed in tissue sections of metastatic specimensderived from 3 different patients with malignant melanoma (FIGS. 9a and9 c-d), but not from a non-malignant nevus (FIG. 9b). Altogether, theseresults imply a potential use of hpa specific primers, nucleic acidprobes and antibodies in early diagnosis of melanoma metastasis.

[0142] Human liver carcinoma: The heparanase enzyme was first purifiedin our laboratory from a human hepatoma cell line (Sk-Hep-1). In fact,amino acid sequences derived from the purified hepatoma heparanase wereused to clone the hpa gene. In situ hybridization studies revealed anintense expression of the hpa gene in tissue sections derived from humanheaptocellular carcinoma (FIGS. 10a-b) and liver adenocarcinoma (FIG.10c). The hpa mRNA was not expressed by adult normal liver tissue (FIG.10d). It was expressed, however, in embryonic human liver (FIG. 10e).Each of these examples clearly supports the use of heparanase specificmolecular probes as tools for early diagnosis of human cancer and itsspread and response to anti-cancer treatments.

[0143] Other human tumors: A preferential expression of the hpa gene wasclearly observed by in situ hybridization performed with biopsyspecimens of several different human carcinomas in comparison with theirnormal tissue counterparts. As demonstrated in FIGS. 11a-f, an intenseexpression of the hpa gene was observed in tissue sections derived fromadenocarcinoma of the ovary (FIG. 11a), squameous cell carcinoma of thecervix (FIG. 11c), and colon adenocarcinoma (FIG. 11e). In contrast,there was little or no expression of the hpa mRNA in human tissuesections derived from normal ovary (FIG. 11b), cervix (FIG. 11d) andsmall intestine (FIG. 11f). The few cells stained in the normal tissuespecimens were single infiltrating macrophages and neutrophils.

[0144] Positive staining of the hpa gene was also clearly seen inadenocarcinoma of the stomach (FIG. 12a), teratocarcinoma (FIG. 12b),well differentiated endometrial adenocarcinoma (FIG. 12c),adenocarcinoma of the pancreas (FIG. 12d), and mesothelioma (FIG. 12e).Each of these examples clearly supports the use of heparanase specificmolecular probes as tools for early diagnosis of human cancer and itsspread and response to anti-cancer treatments.

[0145] Human leukemia and lymphoma: We have previously applied timeconsuming measurements of heparanase activity and demonstrated thatheparanase is expressed and readily secreted by acute and chronic humanmyeloid leukemic cells (AML and CML), but not by chronic lymphocyticleukemic cells (CLL). The availability of heparanase specific primersenables a more sensitive and rapid determination of hpa gene expressionby human leukemia and lymphoma cells. For this purpose, peripheral whiteblood cells (derived from patients with leukemia and lymphoma) werepurified on Ficoll-hypack and subjected to total RNA isolation andRT-PCR determination of the hpa mRNA. Altogether, cells of 69 patientswere tested. Representative patients are presented in FIGS. 13a-b andthe results are summarized in Table 1 below. Cells from 31 out of 31patients with CLL showed no detectable expression of the hpa gene (FIG.13a, lanes 1-5, FIG. 13b, lanes 2, 7, 10 and 12) regardless of the stageof the disease. Similar results were obtained with cells from 4 out of 4patients with non-Hodjkin lymphoma (NHL) (FIG. 13b, lanes 5 and 6). Boththe CLL and NHL cells represent primarily differentiated B cells. Incontrast, the hpa mRNA was expressed by cells derived from 14 out of 14patients with AML (FIG. 13b, lane 11). These cells representundifferentiated myeloblasts of neutrophils and monocyte origin. The hpamRNA was expressed in cells of 1 out of 3 patients with CML, and 2 outof 2 patients with acute lymphocytic leukemia. Surprisingly, umbilicalcord blood derived white blood cells showed little (one case) or noexpression (13 additional cases) of the hpa gene in different cord bloodsamples (FIG. 14, Table 1, below). These cord blood preparations areenriched with hematopoietic stem cells. Studies with established celllines (FIG. 15) revealed no expression of the hpa mRNA in Burkitt Blymphoma (i.e., Raji, Daudi, DG-75, lanes 2-4, respectively), as opposedto mature normal B (Ebv transformed) lymphoblastoid cell line (i.e.,monga, FIG. 15, lane 1) and erythroleukemia (K-562, lane 5).

[0146] Apparently, heparanase expression can distinguish betweendifferentiated B cell lymphoma (CLL and NHL) and undifferentiatedmyelocytic and lymphoblastoid leukemia (AML and ALL) (Table 1). The lackof hpa gene expression by umbilical cord white blood cells may enable todistinguish between early normal white blood cells (hpa negative) andearly leukemic cells (hpa positive). Furthermore, the presence ofheparanase may distinguish between early lymphatic leukemic cells (hpapositive) and late B leukemia and lymphoma cells (hpa negative). TABLE 1Expression of hpa mRNA (RT-PCR) in human leukemia, lymphoma and melanomaType # of patients # hpa positive # hpa negative CLL 31 0 31 AML 14 14 0ALL 2 2 0 CML 3 1 2 NHL 4 0 4 Cord blood 14 1 13 Melanoma 16 16 0 Nevus(normal) 3 0 3

[0147] Heparanase activity in the urine of cancer patients: In anattempt to elucidate the involvement of heparanase in tumor progressionand its relevance to human cancer, we screened urine samples forheparanase activity. Heparanase activity was determined by incubation ofurine with soluble sulfate labeled proteoglycans obtained by trypsindigestion of metabolically Na₂ ³⁵SO₄ labeled subendothelialextracellular matrix. Heparanase activity resulted in conversion of ahigh molecular weight (MW) sulfate labeled substrate into low MW heparansulfate degradation fragments as determined by gel filtration analysis.Heparanase activity was detected in the urine of 21 (renal cellcarcinoma, breast carcinoma, rabdomyosarcoma, stomach cancer, myeloma)out of 157 cancer patients. Three examples are given in FIGS. 16a-c.High levels of heparanase activity were determined in the urine ofpatients with an aggressive disease (primarily breast carcinoma, FIGS.16b-c, multiple myeloma, FIG. 16a) and there was no detectable activityin the urine of healthy donors (FIG. 16d). A more sensitive ELISA isexpected to detect the heparanase protein at early stages of thedisease. Urine may also contain heparanase inhibitors (i.e., GAGs) andhence an activity assay may under estimate the number of patients withpositive urinary heparanase protein.

[0148] Heparanase activity in the urine of diabetic patients: Reductionin glomerular basement membrane (GBM) heparan sulfate proteoglycan(HSPG) is responsible for the microalbuminuria and proteinuria ofdiabetic nephropathy. We identified heparanase activity in cultured ratmesangial cells and postulated that the reduction in glomerular HSPG issecondary to increased glomerular heparanase activity and that thelatter will be manifested by an increase in urinary heparanase. Urinaryheparanase activity was tested in samples from 70 patients with type Idiabetes and in 40 sex and age matched controls, as described above. Theresults are summarized in Table 2 below. Fifty patients werenormoalbuminuric (NA) while 20 had microalbuminuria (MA). Urinaryheparanase activity was detected in 13 of 70 (19%) diabetic patientswhile it was absent in the control group (p=0.002). Sixteen percent ofthe NA patients and 25% of the MA patients showed urinary heparanaseactivity (FIGS. 16g-h). Interestingly, over 80% of the heparanasepositive patients were females. Heparanase positive patients hadsignificantly higher blood glucose (p=0.0005) and HbA1C (p=0.03) levelscompared with heparanase negative diabetic patients. This is the firststudy suggesting a role for heparanase in the pathogenesis of diabeticnephropathy. Urinary heparanase may be an early marker for renalinvolvement in type I diabetic patients, anteceding MA. The presence ofheparanase activity in the urine of normo and microalbuminuric IDDM(insulin dependent diabetic mellitus) patients, is most likely due todiabetic nephropathy, the most important single disorder leading torenal failure in adults. TABLE 2 Heparanase activity in urine of IDDMpatients No. of Averaged Disease Heparanase patients Age Sex durationBlood pres GFR positive Normo- 50 26.2 ± 8.5 26 males 16.5 ± 7.3 112 ±17 134 ± 25 8/50 (16%) albuminuria years 24 females years ml/min/1.73 m²Micro- 20 26.5 ± 11.2 10 males 14.5 ± 7.9 115 ± 13 128 ± 26 5/20 (25%)albuminuria years 10 females years ml/min/1.73 m²

[0149] Repeated determination of urinary heparanase in 9 IDDM patientsyielded similar results (6 negative and 3 positive) to the initialanalysis performed 3 months earlier. Our results suggest that heparanaseactivity may play a role in the regulation of the number of HSPG anionicsites in the GBM and hence may modulate the permselective properties ofthe glomerular basement membrane.

[0150] Heparan sulfate contributes to the assembly and integrity of theECM through binding to various ECM molecules such as collagen, laminin,fibronectin, thrombospondin and tenascin. Cleavage of heparan sulfatemay therefore result in disassembly of the ECM leading to a loss of itsbarrier properties. We have identified heparanase activity expressed bymesanglial cells (not shown). Once heparanase is secreted by stimulatedmesangial cells it will degrade heparan sulfate in the GBM thus allowingits passage into the urinary space.

[0151] Heparanase activity was also detected in the urine of proteinuricpatients not suffering from diabetes (FIGS. 16e-f). These includedpatients with focal segmental glomerulosclerosis, minimal changenephrotic syndrome and congenital nephrotic syndrome, thus indicatingthat the involvement of heparanase in the generation of proteinuria maynot be limited to diabetic nephropathy. Urinary heparanase activityseems to be detected more frequently as the degree of proteinuriaincreases. Active heparanase was detected in the urine of 15% ofnormoalbuminuric and 25% microalbuminuric type I diabetic patients. Theprevalence reached 48% in a group of 28 macroalbuminuric patients withNIDDM.

[0152] Diabetic nephropathy, occurring in approximately 30% of patientswith type I diabetes, is a major cause of end stage renal disease. Theinability to discriminate the subpopulation that will develop renaldamage prior to the appearance of microalbuminuria, 10-15 yearsfollowing the diagnosis of diabetes, prevents us from significantlychanging the devastating natural history of the disease. Urinaryheparanase activity is a distinguishing feature, occurring in 30-35% ofnormoalbuminuric females, within an otherwise homogenous group ofpatients.

[0153] This is the first result suggesting a role for heparanase in thepathogenesis of proteinuria in type I diabetes. Obviously, measurementsof urinary heparanase activity is both time consuming and not sensitiveenough. Moreover, we have demonstrated the presence of an inhibitor ofmammalian heparanase in the urine of normal individuals. The nature ofthis inhibitory substance, possibly urinary glycosaminoglycans iscurrently being studied. Urinary heparanase activity is therefore theresult of a balance between the presence in the urine of the enzyme andits inhibitor(s). Immunodetection of the heparanase protein is thereforea more sensitive and straightforward approach for diagnostic purposes.Altogether, our results clearly indicate that anti-heparanase antibodiesthat identify the heparanase antigen can be applied for early diagnosisof cancer metastasis and renal diseases. As discussed above, it isconceivable that heparanase may overcome the filtration barrier of theglomerular basement membrane and ECM simply by virtue of its ability todegrade the HS moieties that are held responsible for theirpermeaselective properties. Urinary heparanase is therefore expected toreflect the presence of heparanase in the circulation and hence be asensitive marker for metastatic, inflammatory and kidney disease. Ofparticular significance is the potential ability to follow the course oftumor progression and spread, response to anti-cancer treatments, andpossible relapse of the disease in a given patient. Targeted drugdelivery and therapy are another aspect of the use for such antibodies.

[0154] Anti-heparanase polyclonal antibodies: Antisera from twoimmunized rabbits were examined by western blot for reactivity withvarious segments of recombinant heparanase expressed in E. coli and withthe Baculovirus expressed heparanase (FIGS. 17a-b). In both cases, thepolyclonal antibody recognized proteins of the expected size in E. coliderived recombinant heparanase, about 60 kDa for the entire open readingframe (lanes 2), about 45 kDa for the 414 amino acids BamHI-KpnI hpafragment (lanes 3) and 35 kDa for the 302 amino acids encoded by aBamHI-HindIII hpa fragment (lanes 4). A protein of approximately 65 kDawas recognized in the medium of Sf21 insect cells infected withrecombinant Baculovirus pFhpa (lanes 7).

[0155] The specificity of affinity purified polyclonal antibodies wasdetermined by Western blot with recombinant heparanase expressed invarious expression systems, baculovirus infected insect cells, the yeastPichia pastoris and CHO cells transfected with the hpa cDNA. For detailsabout the CHO and Pichia clones see U.S. patent application Ser. No.09/071,618, which is incorporated by reference as if fully set forthherein.

[0156] The specificity of the purified antibody is demonstrated in FIG.18. The purified antibody identified a single about 65 kDa proteinexpressed by Pichia pastoris (FIG. 18, lane 4), and a major band ofsimilar size expressed by Sf21 cells infected with recombinantbaculovirus (FIG. 18, lane 1). In a CHO stable transfected clone, 65 kDaand 50 kDa bands are detected (FIG. 18, lane 3) as compared with thenegative control (FIG. 18, lane 2). In several experiments the two formsof the recombinant heparanase were identified, the higher form appearedas 60 to 65 kDa and the lower form as 45 to 50 kDa. Antibody 7644 wasmore specific and detected mainly the bands of the recombinantheparanase. 7460 detected several other cross reactive bands.

[0157] As shown in FIG. 19a, crude polyclonal antibodies recognizedmultiple bands in human platelets (lanes 2 and 3) and neutrophils cellextracts (lanes 4 and 5), as well as mouse melanoma cell line B16 (lanes6 and 7). However, as shown in FIG. 19b, affinity purified antibodiesrecognized the 65 kDa and 50 kDa forms of heparanase purified fromplacenta (lane 1), two major bands in platelets extract, an upper bandof approximately 50 kDa which corresponds with the lower band of thepurified protein and a lower band of about 30 kDa (lanes 2 and 3). The50 kDa protein appears in mouse melanoma cells as well as two bands of ahigher molecular weight and several minor bands, which represent crossreactive proteins or other species of heparanase (lanes 6 and 7).

[0158] Monoclonal antibodies: Eight hundreds hybridomas, generatedfollowing 3 fusions were screened by ELISA for reactivity against humanheparanase (native and denatured). Eight positive hybridomas wereselected. Table 3 below summarizes the characteristics of the 8hybridomas. TABLE 3 Relative reactivity of hybridomas supernatants withnative and denatured recombinant human heparanase ELISA Hybridoma NativeDenature Western blotting HP-6 − + n.d. HP-40 +++ ++ n.d. HP-45 + ++n.d. HP-92 ++ +++ n.d/ HP-117 ++++ +++ 60,45,42 kDa HP-130 ++++ +++ n.d.HP-239 ++++ ++ n.d. HP-303 − ++ n.d.

[0159] Immunoblot of native and recombinant heparanase expressed invarious cell types was performed using the supernatant of hybridomaHP-117 (FIG. 20). A major band of approximately 50 kDa was detected inextract of stably transfected CHO cells (lane 3) and in plateletsextract (lane 6). This band is also detected in transfected 293 cells ascompared to the negative control (lanes 2 and 1 respectively). A band ofapproximately 42 kDa was observed in all mammalian cell extracts,including the negative control. This band probably represent a crossreactive protein or an endogenous form of heparanase. The 65 kDarecombinant heparanase purified from medium of baculovirus infectedinsect cells is clearly observed in lane 5 as well as a band of 53 kDain lane 6 which is the expected size of the 508 amino acids heparanasepolypeptide expressed in the E. coli. cells.

[0160] Both polyclonal and monoclonal antibodies were used successfullyfor detection of heparanase in intact cells by immunohistochemistry.Polyclonal antibodies showed specific staining of CHO cells transfectedwith pShpaCdhfr expression vector as described in patent U.S. patentapplication Ser. No. 09/071,618, which is incorporated by reference asif fully set forth herein, as compared with no staining of thenon-transfected CHO cells (FIGS. 21a-b). Similar results were obtainedwith several monoclonal antibodies. FIGS. 22a-b demonstrate the specificstaining of heparanase in the cytoplasm of transfected CHO cells, withsupernatant of hybridoma HP-130. No staining was observed innon-transfected cells. Monoclonal antibody HP-92 showed a specificstaining of neutrophils and platelets in blood smear of a healthy donor(FIGS. 23a-c). This expression pattern is consistent with the highlevels of heparanase activity characteristic of these cells.

[0161] Availability of anti-heparanase antibodies will enabledevelopment of immunological assays for screening tissue and body fluidsfor heparanase. An ELISA will provide a more sensitive and convenientmeans of detection as compared to the currently available assays ofheparanase activity which do not appear sensitive enough for thedetection of the enzyme in non-concentrated plasma and body fluids.

[0162] ELISA will provide a powerful diagnostic tool for quantitativedetermination of heparanase concentrations in serum, plasma, urine andother biological fluids.

[0163] Although platelets and activated cells of the immune system (11)can express heparanase activity under certain conditions, we havedetected little or no heparanase activity in normal human plasma. Thepossibility arises that with cancer patients, particularly those withleukemia and lymphoma, heparanase is secreted into the blood stream. Infact, our studies indicate that both acute and chronic human myeloidleukemic cells (AML and CML), but not chronic lymphocytic leukemic cells(CLL), secrete substantial amounts of heparanase during short incubationin PBS at 4° C.

[0164] As described above, elevated levels of heparanase were detectedin sera from metastatic tumor bearing animals and melanoma patients (13)and in tumor biopsies of cancer patients (15). High levels of heparanaseactivity were measured in the urine of patients with aggressivemetastatic disease and there was no detectable activity in the urine ofhealthy donors.

[0165] Although the invention has been described in conjunction withspecific embodiments thereof, it is evident that many alternatives,modifications and variations will be apparent to those skilled in theart. Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

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1 7 1 1721 DNA Homo sapiens 1 ctagagcttt cgactctccg ctgcgcggcagctggcgggg ggagcagcca ggtgagccca 60 agatgctgct gcgctcgaag cctgcgctgccgccgccgct gatgctgctg ctcctggggc 120 cgctgggtcc cctctcccct ggcgccctgccccgacctgc gcaagcacag gacgtcgtgg 180 acctggactt cttcacccag gagccgctgcacctggtgag cccctcgttc ctgtccgtca 240 ccattgacgc caacctggcc acggacccgcggttcctcat cctcctgggt tctccaaagc 300 ttcgtacctt ggccagaggc ttgtctcctgcgtacctgag gtttggtggc accaagacag 360 acttcctaat tttcgatccc aagaaggaatcaacctttga agagagaagt tactggcaat 420 ctcaagtcaa ccaggatatt tgcaaatatggatccatccc tcctgatgtg gaggagaagt 480 tacggttgga atggccctac caggagcaattgctactccg agaacactac cagaaaaagt 540 tcaagaacag cacctactca agaagctctgtagatgtgct atacactttt gcaaactgct 600 caggactgga cttgatcttt ggcctaaatgcgttattaag aacagcagat ttgcagtgga 660 acagttctaa tgctcagttg ctcctggactactgctcttc caaggggtat aacatttctt 720 gggaactagg caatgaacct aacagtttccttaagaaggc tgatattttc atcaatgggt 780 cgcagttagg agaagattat attcaattgcataaacttct aagaaagtcc accttcaaaa 840 atgcaaaact ctatggtcct gatgttggtcagcctcgaag aaagacggct aagatgctga 900 agagcttcct gaaggctggt ggagaagtgattgattcagt tacatggcat cactactatt 960 tgaatggacg gactgctacc agggaagattttctaaaccc tgatgtattg gacattttta 1020 tttcatctgt gcaaaaagtt ttccaggtggttgagagcac caggcctggc aagaaggtct 1080 ggttaggaga aacaagctct gcatatggaggcggagcgcc cttgctatcc gacacctttg 1140 cagctggctt tatgtggctg gataaattgggcctgtcagc ccgaatggga atagaagtgg 1200 tgatgaggca agtattcttt ggagcaggaaactaccattt agtggatgaa aacttcgatc 1260 ctttacctga ttattggcta tctcttctgttcaagaaatt ggtgggcacc aaggtgttaa 1320 tggcaagcgt gcaaggttca aagagaaggaagcttcgagt ataccttcat tgcacaaaca 1380 ctgacaatcc aaggtataaa gaaggagatttaactctgta tgccataaac ctccataacg 1440 tcaccaagta cttgcggtta ccctatcctttttctaacaa gcaagtggat aaataccttc 1500 taagaccttt gggacctcat ggattactttccaaatctgt ccaactcaat ggtctaactc 1560 taaagatggt ggatgatcaa accttgccacctttaatgga aaaacctctc cggccaggaa 1620 gttcactggg cttgccagct ttctcatatagtttttttgt gataagaaat gccaaagttg 1680 ctgcttgcat ctgaaaataa aatatactagtcctgacact g 1721 2 543 PRT Homo sapiens 2 Met Leu Leu Arg Ser Lys ProAla Leu Pro Pro Pro Leu Met Leu Leu 1 5 10 15 Leu Leu Gly Pro Leu GlyPro Leu Ser Pro Gly Ala Leu Pro Arg Pro 20 25 30 Ala Gln Ala Gln Asp ValVal Asp Leu Asp Phe Phe Thr Gln Glu Pro 35 40 45 Leu His Leu Val Ser ProSer Phe Leu Ser Val Thr Ile Asp Ala Asn 50 55 60 Leu Ala Thr Asp Pro ArgPhe Leu Ile Leu Leu Gly Ser Pro Lys Leu 65 70 75 80 Arg Thr Leu Ala ArgGly Leu Ser Pro Ala Tyr Leu Arg Phe Gly Gly 85 90 95 Thr Lys Thr Asp PheLeu Ile Phe Asp Pro Lys Lys Glu Ser Thr Phe 100 105 110 Glu Glu Arg SerTyr Trp Gln Ser Gln Val Asn Gln Asp Ile Cys Lys 115 120 125 Tyr Gly SerIle Pro Pro Asp Val Glu Glu Lys Leu Arg Leu Glu Trp 130 135 140 Pro TyrGln Glu Gln Leu Leu Leu Arg Glu His Tyr Gln Lys Lys Phe 145 150 155 160Lys Asn Ser Thr Tyr Ser Arg Ser Ser Val Asp Val Leu Tyr Thr Phe 165 170175 Ala Asn Cys Ser Gly Leu Asp Leu Ile Phe Gly Leu Asn Ala Leu Leu 180185 190 Arg Thr Ala Asp Leu Gln Trp Asn Ser Ser Asn Ala Gln Leu Leu Leu195 200 205 Asp Tyr Cys Ser Ser Lys Gly Tyr Asn Ile Ser Trp Glu Leu GlyAsn 210 215 220 Glu Pro Asn Ser Phe Leu Lys Lys Ala Asp Ile Phe Ile AsnGly Ser 225 230 235 240 Gln Leu Gly Glu Asp Tyr Ile Gln Leu His Lys LeuLeu Arg Lys Ser 245 250 255 Thr Phe Lys Asn Ala Lys Leu Tyr Gly Pro AspVal Gly Gln Pro Arg 260 265 270 Arg Lys Thr Ala Lys Met Leu Lys Ser PheLeu Lys Ala Gly Gly Glu 275 280 285 Val Ile Asp Ser Val Thr Trp His HisTyr Tyr Leu Asn Gly Arg Thr 290 295 300 Ala Thr Arg Glu Asp Phe Leu AsnPro Asp Val Leu Asp Ile Phe Ile 305 310 315 320 Ser Ser Val Gln Lys ValPhe Gln Val Val Glu Ser Thr Arg Pro Gly 325 330 335 Lys Lys Val Trp LeuGly Glu Thr Ser Ser Ala Tyr Gly Gly Gly Ala 340 345 350 Pro Leu Leu SerAsp Thr Phe Ala Ala Gly Phe Met Trp Leu Asp Lys 355 360 365 Leu Gly LeuSer Ala Arg Met Gly Ile Glu Val Val Met Arg Gln Val 370 375 380 Phe PheGly Ala Gly Asn Tyr His Leu Val Asp Glu Asn Phe Asp Pro 385 390 395 400Leu Pro Asp Tyr Trp Leu Ser Leu Leu Phe Lys Lys Leu Val Gly Thr 405 410415 Lys Val Leu Met Ala Ser Val Gln Gly Ser Lys Arg Arg Lys Leu Arg 420425 430 Val Tyr Leu His Cys Thr Asn Thr Asp Asn Pro Arg Tyr Lys Glu Gly435 440 445 Asp Leu Thr Leu Tyr Ala Ile Asn Leu His Asn Val Thr Lys TyrLeu 450 455 460 Arg Leu Pro Tyr Pro Phe Ser Asn Lys Gln Val Asp Lys TyrLeu Leu 465 470 475 480 Arg Pro Leu Gly Pro His Gly Leu Leu Ser Lys SerVal Gln Leu Asn 485 490 495 Gly Leu Thr Leu Lys Met Val Asp Asp Gln ThrLeu Pro Pro Leu Met 500 505 510 Glu Lys Pro Leu Arg Pro Gly Ser Ser LeuGly Leu Pro Ala Phe Ser 515 520 525 Tyr Ser Phe Phe Val Ile Arg Asn AlaLys Val Ala Ala Cys Ile 530 535 540 3 1721 DNA Homo sapiens CDS(63)..(1691) 3 ctagagcttt cgactctccg ctgcgcggca gctggcgggg ggagcagccaggtgagccca 60 ag atg ctg ctg cgc tcg aag cct gcg ctg ccg ccg ccg ctg atgctg 107 Met Leu Leu Arg Ser Lys Pro Ala Leu Pro Pro Pro Leu Met Leu 1 510 15 ctg ctc ctg ggg ccg ctg ggt ccc ctc tcc cct ggc gcc ctg ccc cga155 Leu Leu Leu Gly Pro Leu Gly Pro Leu Ser Pro Gly Ala Leu Pro Arg 2025 30 cct gcg caa gca cag gac gtc gtg gac ctg gac ttc ttc acc cag gag203 Pro Ala Gln Ala Gln Asp Val Val Asp Leu Asp Phe Phe Thr Gln Glu 3540 45 ccg ctg cac ctg gtg agc ccc tcg ttc ctg tcc gtc acc att gac gcc251 Pro Leu His Leu Val Ser Pro Ser Phe Leu Ser Val Thr Ile Asp Ala 5055 60 aac ctg gcc acg gac ccg cgg ttc ctc atc ctc ctg ggt tct cca aag299 Asn Leu Ala Thr Asp Pro Arg Phe Leu Ile Leu Leu Gly Ser Pro Lys 6570 75 ctt cgt acc ttg gcc aga ggc ttg tct cct gcg tac ctg agg ttt ggt347 Leu Arg Thr Leu Ala Arg Gly Leu Ser Pro Ala Tyr Leu Arg Phe Gly 8085 90 95 ggc acc aag aca gac ttc cta att ttc gat ccc aag aag gaa tca acc395 Gly Thr Lys Thr Asp Phe Leu Ile Phe Asp Pro Lys Lys Glu Ser Thr 100105 110 ttt gaa gag aga agt tac tgg caa tct caa gtc aac cag gat att tgc443 Phe Glu Glu Arg Ser Tyr Trp Gln Ser Gln Val Asn Gln Asp Ile Cys 115120 125 aaa tat gga tcc atc cct cct gat gtg gag gag aag tta cgg ttg gaa491 Lys Tyr Gly Ser Ile Pro Pro Asp Val Glu Glu Lys Leu Arg Leu Glu 130135 140 tgg ccc tac cag gag caa ttg cta ctc cga gaa cac tac cag aaa aag539 Trp Pro Tyr Gln Glu Gln Leu Leu Leu Arg Glu His Tyr Gln Lys Lys 145150 155 ttc aag aac agc acc tac tca aga agc tct gta gat gtg cta tac act587 Phe Lys Asn Ser Thr Tyr Ser Arg Ser Ser Val Asp Val Leu Tyr Thr 160165 170 175 ttt gca aac tgc tca gga ctg gac ttg atc ttt ggc cta aat gcgtta 635 Phe Ala Asn Cys Ser Gly Leu Asp Leu Ile Phe Gly Leu Asn Ala Leu180 185 190 tta aga aca gca gat ttg cag tgg aac agt tct aat gct cag ttgctc 683 Leu Arg Thr Ala Asp Leu Gln Trp Asn Ser Ser Asn Ala Gln Leu Leu195 200 205 ctg gac tac tgc tct tcc aag ggg tat aac att tct tgg gaa ctaggc 731 Leu Asp Tyr Cys Ser Ser Lys Gly Tyr Asn Ile Ser Trp Glu Leu Gly210 215 220 aat gaa cct aac agt ttc ctt aag aag gct gat att ttc atc aatggg 779 Asn Glu Pro Asn Ser Phe Leu Lys Lys Ala Asp Ile Phe Ile Asn Gly225 230 235 tcg cag tta gga gaa gat tat att caa ttg cat aaa ctt cta agaaag 827 Ser Gln Leu Gly Glu Asp Tyr Ile Gln Leu His Lys Leu Leu Arg Lys240 245 250 255 tcc acc ttc aaa aat gca aaa ctc tat ggt cct gat gtt ggtcag cct 875 Ser Thr Phe Lys Asn Ala Lys Leu Tyr Gly Pro Asp Val Gly GlnPro 260 265 270 cga aga aag acg gct aag atg ctg aag agc ttc ctg aag gctggt gga 923 Arg Arg Lys Thr Ala Lys Met Leu Lys Ser Phe Leu Lys Ala GlyGly 275 280 285 gaa gtg att gat tca gtt aca tgg cat cac tac tat ttg aatgga cgg 971 Glu Val Ile Asp Ser Val Thr Trp His His Tyr Tyr Leu Asn GlyArg 290 295 300 act gct acc agg gaa gat ttt cta aac cct gat gta ttg gacatt ttt 1019 Thr Ala Thr Arg Glu Asp Phe Leu Asn Pro Asp Val Leu Asp IlePhe 305 310 315 att tca tct gtg caa aaa gtt ttc cag gtg gtt gag agc accagg cct 1067 Ile Ser Ser Val Gln Lys Val Phe Gln Val Val Glu Ser Thr ArgPro 320 325 330 335 ggc aag aag gtc tgg tta gga gaa aca agc tct gca tatgga ggc gga 1115 Gly Lys Lys Val Trp Leu Gly Glu Thr Ser Ser Ala Tyr GlyGly Gly 340 345 350 gcg ccc ttg cta tcc gac acc ttt gca gct ggc ttt atgtgg ctg gat 1163 Ala Pro Leu Leu Ser Asp Thr Phe Ala Ala Gly Phe Met TrpLeu Asp 355 360 365 aaa ttg ggc ctg tca gcc cga atg gga ata gaa gtg gtgatg agg caa 1211 Lys Leu Gly Leu Ser Ala Arg Met Gly Ile Glu Val Val MetArg Gln 370 375 380 gta ttc ttt gga gca gga aac tac cat tta gtg gat gaaaac ttc gat 1259 Val Phe Phe Gly Ala Gly Asn Tyr His Leu Val Asp Glu AsnPhe Asp 385 390 395 cct tta cct gat tat tgg cta tct ctt ctg ttc aag aaattg gtg ggc 1307 Pro Leu Pro Asp Tyr Trp Leu Ser Leu Leu Phe Lys Lys LeuVal Gly 400 405 410 415 acc aag gtg tta atg gca agc gtg caa ggt tca aagaga agg aag ctt 1355 Thr Lys Val Leu Met Ala Ser Val Gln Gly Ser Lys ArgArg Lys Leu 420 425 430 cga gta tac ctt cat tgc aca aac act gac aat ccaagg tat aaa gaa 1403 Arg Val Tyr Leu His Cys Thr Asn Thr Asp Asn Pro ArgTyr Lys Glu 435 440 445 gga gat tta act ctg tat gcc ata aac ctc cat aacgtc acc aag tac 1451 Gly Asp Leu Thr Leu Tyr Ala Ile Asn Leu His Asn ValThr Lys Tyr 450 455 460 ttg cgg tta ccc tat cct ttt tct aac aag caa gtggat aaa tac ctt 1499 Leu Arg Leu Pro Tyr Pro Phe Ser Asn Lys Gln Val AspLys Tyr Leu 465 470 475 cta aga cct ttg gga cct cat gga tta ctt tcc aaatct gtc caa ctc 1547 Leu Arg Pro Leu Gly Pro His Gly Leu Leu Ser Lys SerVal Gln Leu 480 485 490 495 aat ggt cta act cta aag atg gtg gat gat caaacc ttg cca cct tta 1595 Asn Gly Leu Thr Leu Lys Met Val Asp Asp Gln ThrLeu Pro Pro Leu 500 505 510 atg gaa aaa cct ctc cgg cca gga agt tca ctgggc ttg cca gct ttc 1643 Met Glu Lys Pro Leu Arg Pro Gly Ser Ser Leu GlyLeu Pro Ala Phe 515 520 525 tca tat agt ttt ttt gtg ata aga aat gcc aaagtt gct gct tgc atc 1691 Ser Tyr Ser Phe Phe Val Ile Arg Asn Ala Lys ValAla Ala Cys Ile 530 535 540 tgaaaataaa atatactagt cctgacactg 1721 4 26DNA Artificial sequence Single strand DNA oligonucleotide 4 cgcatatgcaggacgtcgtg gacctg 26 5 24 DNA Artificial sequence Single strand DNAoligonucleotide 5 tatgatcctc tagtacttct cgac 24 6 23 DNA Artificialsequence Single strand DNA oligonucleotide 6 ttcgatccca agaaggaatc aac23 7 24 DNA Artificial sequence Single strand DNA oligonucleotide 7gtagtgatgc catgtaactg aatc 24

What is claimed is:
 1. An oligonucleotide comprising a nucleic acidsequence specifically hybridizable with heparanase encoding nucleicacid.
 2. An antisense nucleic acid molecule comprising a nucleic acidsequence specifically hybridizable with heparanase messenger RNA.
 3. Asense nucleic acid molecule comprising a nucleic acid sequencespecifically hybridizable with heparanase antisense RNA.
 4. A pair ofpolymerase chain reaction primers comprising a sense primer and anantisense primers, each of said primers including a nucleic acidsequence specifically hybridizable with heparanase encoding nucleicacid.